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Lab Manual

Lab Manual

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Sections

  • THE GRAM STAIN
  • THE ENDOSPORE STAIN
  • THE ACID-FAST STAIN
  • THE CAPSULE STAIN
  • TRANSFER OF BACTERIA USING ASEPTIC TECHNIQUE
  • PURE CULTURE TECHNIQUES
  • BACTERIAL COLONY MORPHOLOGY
  • COUNTING BACTERIA
  • EVALUATION OF ANTIMICROBIAL CHEMICALS
  • KIRBY-BAUER TEST FOR ANTIBIOTIC SUSCEPTIBILITY
  • IDENTIFICATION OF UNKNOWN BACTERIA
  • OXYGEN REQUIREMENTS & CULTURING ANAEROBIC BACTERIA
  • CARBOHYDRATE UTILIZATION
  • CASEIN HYDROLYSIS
  • GELATIN HYDROLYSIS
  • GENUS STAPHYLOCOCCUS: Isolation and Identification
  • GENUS STREPTOCOCCUS: Isolation and Identification
  • GLOSSARY OF TERMS

RICHLAND COLLEGE Jackie Reynolds Mark Farinha

BIOLOGY 2420
LAB MANUAL
MICROBIOLOGY

                       

TABLE OF CONTENTS
Study guide for lab quizzes/practicals

page number
1. Mock Epidemic MICROSCOPY 2. Use of the Microscope 3. Preparation of Specimens 4. Ectoparasites 5. Helminths 6. Ubiquity of Bacteria 7. Fungi 8. Protozoa STAINING TECHNIQUES 9. Simple Stain and Smear Preparation 10. Gram Stain 11. Endospore Stain 12. Acid-Fast Stain 13. Capsule Stain MICROBIAL GROWTH and CONTROL OF GROWTH 14. Transfer of Bacteria Using Aseptic Technique 15. Pure Culture Technique 16. Bacterial Colony Morphology 17. Pipetting and Dilutions 18. Counting Bacteria 19. Environmental Conditions and Bacterial Growth 20. Effects of Temperature on Bacterial Growth 21. Evaluation of Antimicrobial Chemicals 22. Kirby-Bauer Test for Antibiotic Susceptibility 23. Surgical Hand Scrub 24. Bacteriophages BIOCHEMICAL TESTS and UNKNOWN IDENTIFICATION 25. Identification of Unknown Bacteria 26. Dichotomous Key for identification of Bacteria 27. Motility Tests 28. Oxygen Requirements and Culturing Anaerobic Bacteria 29. Carbohydrate Utilization 30. Casein hydrolysis 31. Catalase Test 32. DNAse Production 33. Decarboxylation and Deamination 34. Gelatin Hydrolysis 35. IMViC Tests 36. Lipid Hydrolysis 37. Litmus Milk Utilization 38. Nitrate Reduction 39. Oxidase test 40. Starch Hydrolysis 41. Urea Hydrolysis 42. API 20E Multitest Identification for Gram – Enteric Rods 43. Genus Staphylococcus: Isolation and Identification 44. Genus Streptococcus: Isolation and Identification 45. Serological Testing : Latex Agglutination (Staph, Strep, and Monospot) Table of Biochemical Tests---FAQs and Study Guide GLOSSARY OF TERMS 1 3 11 15 17 21 25 29 33 37 41 43 45 47 53 59 63 69 79 83 87 91 95 99 103 107 109 113 117 119 121 123 125 129 131 135 137 139 141 143 145 147 151 155 161 165 167

and arrangement why incorrect results . sexual spores for yeasts and molds (not specific spore names) the major classes of fungi . agar slant vs.why they bind to the cell GRAM STAIN reagents & procedure interpretation of reaction. differential stain types of dyes . a stained smear advantages/disadvantages of each ECTOPARASITES examples within the Arachnid and Insect classes differences Spring 2011 resolution (and ways to increase it) magnification HELMINTHS representative groups of worms as distinguished on microscope general features of worms in each phylum: platyhelminthes vs.acidic vs. agar plate transfer methods PURE CULTURE TECHNIQUES procedure for types of isolation techniques–pours and streak plates pros and cons of each technique streak/pour technique and why done that way SIMPLE STAINING & SMEAR PREPARATION simple vs.morphology asexual vs. darkfield. shape.differentiation and examples seen in lab representative groups of fungi as distinguished on microscope PROTOZOA representative protozoa seen in lab. as distinguished on microscope general features of protozoa as a whole and each class UBIQUITY OF BACTERIA shapes of bacteria TRANSFER OF BACTERIA advantages/disadvantages of broth vs. phase-contrast) uses parts and functions total magnification determination features: parfocal lens alignment refraction and oil immersion PREPARATION OF SPECIMENS shapes and arrangements of bacteria how to make a wet mount vs.BIOLOGY 2420 STUDY GUIDE FOR LAB PRACTICALS EXERCISE MICROSCOPY (brightfield. trematodes FUNGI structural differences between yeasts and molds . basic . nematodes the 2 classes of platyhelminthes: cestodes vs.

indole. citrate) litmus milk urea hydrolysis hydrogen sulfide arginine/lysine/ornithine decarboxylases phenylalanine deamination. purpose. and I (with a chart) ANAEROBES ways to culture according to oxygen needs candle jar vs. what atmosphere thioglycollate broth classification of microbes according to oxygen needs BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA For the following tests–the medium used. hanging drops) true vs. VP. TTC motility. SIM. nitrate reduction catalase test oxidase test starch hydrolysis lipid hydrolysis casein hydrolysis . -). reagent used gelatin hydrolysis sugar use (phenol red sugars) IMViC tests (MR. false motility (Brownian movement) example of flagellation types interpretation of motility in TTC media CAPSULE STAINS interpretation PIPETTING & DILUTIONS solving dilution problems use (and reading) of the pipette COUNTING BACTERIA . R.how they work. and osmotic pressure on life effect of hypertonic media on growth halophiles/osmophiles UV LIGHT: mechanism of kill limitations of UV kill EFFECTS OF TEMPERATURE ON GROWTH classification based on optimal temperatures EVALUATION OF ANTIMICROBIALS Zones of inhibition How to determine efficiency of chemicals How to do a hand scrub KIRBY-BAUER TEST FOR ANTIBIOTIC SENSITIVITIES zones of inhibition determination of S. osmotic pressure effects of pH. GasPak jar . interpretation (+ vs.STANDARD PLATE COUNT & TURBIDEMETRY determination of bacterial counts in a specimen use of spectrophotometer (basics of what it is reading) use of a graph to quesstimate bacterial counts ENVIRONMENTAL INFLUENCES: pH.SPORE STAIN reagents & interpretation example of spore formers ACID-FAST STAIN reagents & interpretation example of acid-fast bacteria MOTILITY (flagella stain.

and MONOSPOT basics of the antigen-antibody test purpose of the latex beads interpretation . reagent used hemolytic reactions on blood agar BACTERIOPHAGES the major points in technique and reasoning lytic vs. -). lysogenic infection determination of number of viruses/ml plaques SEROLOGICAL LATEX SLIDE AGGLUTINATION: STAPH.API20E pros and cons uses STAPHYLOCOCCUS ID tests for differentiating the genus Staph and its species For any tests/media–purpose. reagent used hemolytic reactions on blood agar STREPTOCOCCUS ID tests for differentiating the genus Strep and its species For any media/tests run–purpose. interpretation (+ vs. STREP. interpretation (+ vs. -).

Dump extra powder back into dish. HANDSHAKE ROTATIONS: #1 #2 #3 #4 #5 #6 #7 4. MATERIALS: glo-germ powder UV light Plate with talc powder (1 per person) PROCEDURE: 1. USE THE RIGHT HAND ONLY. Go through entire round of handshakes. Write out the transmission pattern for the epidemic. Wash your hands after this exercise. 7. do not touch anything or anyone else. The purpose of using this fluorescent powder is to "see" the transmission of microorganisms. OBJECTIVE: Get to know your classmates and appreciate the ease of disease transmission.MOCK EPIDEMIC Page 1 One person in the lab will have a talcum powder contaminated with GLO GERM POWDER. Wait for the instructor to view your hand under ultraviolet light. GLO GERM is NOT a bacterium and is not harmful. AFTER ALL HANDSHAKES. Collect materials for exercise---1 plate of talcum powder per student. Work powder around in the palm of your right hand WITHOUT using your left hand. BE SURE TO FILL IN THE HANDSHAKE ROTATIONS BELOW: you will need the information to solve the epidemic. You can easily see if you have been "infected" by viewing your hand under ultraviolet light. 2. 5. 3. Record the numbers of those people infected with the fluorescent powder. and see if the glo germ came off of your hands using the UV lamp. making sure that your hand REALLY contacts the other person's hand. 6. Pick up dish with powder in your left hand and dump into your right hand. Solve for the person who started the epidemic. . The instructor will direct handshake rotations.

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without moving the stage. with the arm towards you. HANDLING THE MICROSCOPE: • • Carry it with 2 hands---one on the arm and the other under the base. Be able to switch objective lenses. Bright field is “0”. look at the markings in red on the objective lenses. Therefore. save some fungi. phase-contrast. it will be very difficult to get all of the oil off. there are some microbes which cannot be seen even with a microscope. PH4 for 100X. The last person using the microscope may have left it dirty: it is imperative to begin with clean lenses. do not move back to the 40X lens to focus: oil should never get on this lens. Clean ALL objective lenses and the ocular with lens paper BEFORE you even place a slide on the stage. Place the microscope back into the correct spot in the cabinet. You will be using an assigned light microscope for a variety of lab exercises through the semester. 40X. unless it is an electron microscope.USE OF THE MICROSCOPE Page 3 The microscope is absolutely essential to the microbiology lab: most microorganisms cannot be seen without the aid of a microscope. Learn how to use the microscope effectively. These Nikon microscopes have a revolving condenser. Once oil has been added to the slide. If this happens. one for each of the lenses—PH1 for 10X. using various objective lenses. If you forget. Use lens paper (ONLY) to remove any oil from the 100X lens. 100X) OBJECTIVES: Identify the parts of the microscope and their functions. it is extremely important that you understand how to use the microscope effectively and how to use different types of microscopy----brightfield. darkfield is “DF”. Handle the microscope safely and clean it. and darkfield. • • • Determination of total magnification = ocular lens (10X) X objective lens (10X. Become familiar with the 3 variations of light microscopy. such as the viruses. and phase-contrast is “PH. PH3 for 40X. Explain principles and terms used in microscopy and focusing. and you will have to clean the lens thoroughly.” There are 3 settings of phase-contrast. while focused on the specimen. And. and it is a good idea to wipe the condenser lens also. everything from viewing pond water to identification of your unknown bacterium. with specific settings for the 3 kinds of microscopy: the settings are labeled with white letters that can be seen in the front. and particularly the oil immersion lens. Determine total magnification of the specimen. . of course.

but not for microorganisms. and a stained smear that you have made. Refer to the lab exercise PREPARATION OF SPECIMENS.Page 4 MATERIALS NEEDED: microscope lens paper bibulous paper oil dropper prepared wet mounts or stained smears THE PROCEDURES: You will use a variety of specimens with your microscope----a wet mount. a bought prepared smear. Also. We always have the condenser stage closest to the mechanical stage when viewing microorganisms. Rotate the revolving nosepiece until the low power 10Xobjective lens snaps into place. like worms or insects. Bring the stage all the way up. There is a special knob for the condenser stage under the mechanical stage. using the coarse adjustment knob. PH3 for 40X. . you can move the condenser down to improve light density hitting the specimen. The brightfield condenser has a 0 etched in white. move the iris diaphragm left and right so you can see the effect on the amount of light. 4. Clean all lenses (oculars. 5. 2. The condenser gathers all available light from the lamp and directs it up to the stage. Raise the condenser stage ALL THE WAY UP. BEFORE PUTTING A SLIDE ON THE MICROSCOPE STAGE: 1. Keep an eye on the distance between the slide and the lens to MAKE SURE that you do not crash the lens into the stage. The NIKON microscopes have 3 types of condenser lenses for 3 types of light microscopy: o brightfield (O on condenser) o darkfield (DF on condenser) o phase-contrast (condenser settings: PH 1 for 10X. Your light amount coming up through the condenser is controlled by the iris diaphragm. 8. When viewing largest objects. and lens on condenser) with LENS PAPER. 7. Rotate the condenser so that you see all of the settings (white letters are engraved into the front of the condenser dial). Start with brightfield microscopy ALWAYS. This is where the control knob will stay: do not touch it again. objective lenses. 6. Turn the brightness control knob ALL the way up. PH4 for 100X) 3. and then back off 1/4 of a turn. Find all of the structures on the microscope (diagrams below) being sure that you know their functions.

3. Rotate the 40X lens in place. Swing the 40X objective (high dry) out of the way. and place it in the center of the hole allowing light through the stage. Use the fine adjustment knob to focus clearly. Be sure that you are looking through the binocular head of the microscope with BOTH eyes. it is NOT TOUCHED AGAIN. 5. 3. do NOT GO BACK TO THE 40X OBJECTIVE. and you will have to really clean it to get the oil off. Now look through the oculars. STOP using the coarse adjustment. YOU PROBABLY HAVE OIL RESIDUE ON THE 40x OBJECTIVE. making sure that it snaps into place. 2. and rotate the 100X objective (oil immersion) into place. While looking through the ocular eyepiece. 4. You will want to reduce the light coming through the condenser. All focusing will now be done with the fine adjustment knob. AND NO AMOUNT OF FOCUSING BRINGS THE OBJECT INTO VIEW. 2. IF YOUR FIELD OF VISION IS FUZZY. Do NOT MOVE the focus knobs or the stage knobs. The lens will actually GO INTO THE OIL DROP. probably out of focus. After focusing at the beginning with the coarse adjustment knob. lower the stage SLOWLY using the coarse adjustment knob. Your specimen should still be seen in the field of vision. Place the wet mount or prepared smear on the stage. Use your fine adjustment knob to clarify the objects. Set the ocular lenses to the correct distance for your face (the oculars can be moved apart or closer together for your own needs). Your object should still be in the field of vision. so close your iris diaphragm so that you get better contrast of the specimen.Page 5 9. CHANGING OBJECTIVES: The Nikon lenses are PARFOCAL---the objectives are aligned so that rotation to another lens can be done without major focusing. . since the lens should not touch the slide anyway. 100X MAGNIFICATION: 1. TO VIEW A SPECIMEN: 1. 10. Once you have gone into oil immersion. but 4 times larger now. As soon as you see the specimen. Try to guesstimate where the specimen is located on the slide. and secure it inside of the stage holder. IT HAS TO BE CLEANED WELL WITH LENS PAPER. TO MOVE INTO OIL IMMERSION. These ocular eyepiece lenses are both 10X magnification. The objective will get oil on it. increasing your light with your iris diaphragm lever. and switch over to the fine adjustment knob. Place a single drop of immersion oil on the slide right over where the light is coming through the stage. The 10X can be returned to.

making annoying grating noises. ask instructor for some help. Found the specimen on low power. each lens should “click” into place. Depending on whether you want to use the 10X. If that doesn’t work. so if someone else complains about it being left with oil or a slide on the stage. but rather dirt on slide. Clean with lens paper thoroughly. . Turn the coarse adjustment knob so that the stage is far from the lens. Wind the electrical cord around the cord holder properly. Darkfield and phase-contrast microscopies have particular condenser lenses required for proper visualization. Light off center? Do you have the lens correctly in place? As you rotate the nosepiece. and you will know that it is in the correct position. with specimen facing down towards stage. you can easily switch to another type of microscopy: Just rotate the condenser knob. Once through with the microscope. TROUBLESHOOTING: Focus fuzzy? Probably oil on the lens. Xylene may be used. Remove any slide left on the stage. Be sure that you are using the correct condenser setting for that particular objective lens. you or another person who is assigned that particular scope will be reprimanded. but SPARINGLY since it can destroy the glue seating the lens. although SOMETIMES it is helpful for delineating subtle shapes and colors that cannot be readily seen using brightfield. Be sure that your iris diaphragm is OPEN all the way. If that does not help. using 10X and 40X (100X will not show well). The lens could hit against the stage and get scratched. PLACE YOUR MICROSCOPE BACK IN ITS NUMBERED POSITION IN THE CORRECT CABINET. Do NOT drag it across the table. 40X. use lens cleaner with lens paper. USING DARKFIELD AND PHASE-CONTRAST: Once you are looking at your object using brightfield microscopy. Make sure that the 10X low power lens is in place.Page 6 4. use the lens paper to wipe the oil from the 100X objective lens. pointing towards the stage---not the 100X oil immersion lens. Slide has been turned upside down. HOWEVER: Darkfield is used for wet mounts. Phase-contrast is used for wet mounts also. or 100X objective lens. but lose it when moving into a high power? • • Not focusing on the specimen. PLACING MICROSCOPES BACK INTO THE CABINETS: • • • • • • • YOU are responsible for your assigned microscope! There is only 1 person in each lab who is assigned that particular microscope. you will have to change the phase-contrast consider lens to the appropriate setting.

• Page 7 Specimen off-center on slide: when moving to a higher lens magnification. the specimen is outside the field of vision. .

4.Page 8 PART OF MICROSCOPE 1. stage . 2. coarse adjustment knob fine adjustment knob arm power switch/brightness control base condenser knob iris diaphragm lever FUNCTION general focus. 7. stage holder knobs 13. objective lens 9. 5. or 100X attachment of objective lenses magnification of 10X hold slide in place movement of stage. 40X. stage holder 12. 6. 2 directions placement of slide 8. particularly for 10X lens fine focus. 3. changes intensity infrastructure of the microscope condenser movement control of cone of light coming through condenser magnification of 10X. ocular eyepiece lens 11. revolving nosepiece 10. particularly for 100X lens infrastructure of the microscope on/off switch for light.

What condenser setting value do you want when you are using the 100X objective lens? 5. Which objective lens is also called the oil-immersion lens? 2. What term is used to describe the feature of the microscope that makes it possible to move among the objective lenses with just MINOR focusing? 4. What is parfocal? 8. Why move the 10X objective lens into place when putting the microscope back into the cabinet? 7. How do the functions of the substage condenser and the iris diaphragm within the condenser differ? 3. What would be the total magnification of a specimen using the 40X objective lens? 6.Page 9 LABORATORY REPORT SHEET QUESTIONS: 1. Why reduce your light when using the 10X objective lens? .

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Acid dyes are negatively charged and are repelled by the surface of the cell . You will be staining the bacterial suspension from your mouth. Streptobacillus is seen as a chain of rods. but you can distinguish among shapes. Streptococcus pneumoniae is a coccus found in pairs. Aerococcus is seen as cocci in a foursome. thereby staining the background area rather than the cell itself. also called diplococci. shapes. algae. SHAPES & ARRANGEMENTS OF BACTERIA cocci (coccus = singular) chain pair cluster tetrad bacilli (bacillus = singular) (also called rod) chain spirals Often. However. and arrangements of bacteria. worms and insects (or their larvae) may also be found. having a + charge on the molecule. sizes. crystal violet. Most of the dyes used in the lab are basic dyes. and are attracted to the slight negative charge on the surface of microbial cells. using only one dye. malachite green. being used in the the gram stain. For example: Streptococcus (strepto = chain) is found as a chain of cocci. sizes. or tetrad. You will be looking at living (and often motile) microorganisms of various kinds in pond water--bacteria. safrinin. Although not microorganisms. there is a hint in the name of the microorganism that gives you specific information about shape or arrangement. but still often found in pond water. . bacteria are difficult to see in a wet mount unstained: they are transparent and tiny. and protozoa. Unfortunately. Examples are nigrosine and congo red. and carbol fuschin are all basic dyes. Methylene blue. and acid-fast stain. Everything on the slide will be the same color. Staphylococcus (staphylo = cluster) is seen as cocci in various cluster sizes. and arrangements of cells. spore stain.Page 11 PREPARATION OF SPECIMENS Here is your first exposure to the preparation of a bacterial smear and subsequent staining of it. seeing a variety of bacterial species. you are making a simple stain.

5. Prepare smears of microbial suspensions and stain them. 3. Protozoa will be feeding at the bottom.) toothpicks dye kits THE PROCEDURES: Prepare a wet mount of pond water. with your dropper. Identify the purpose of staining and the uses of a simple stain. Staphylococcus. Go DOWN into the algae and muck to get a really good sample of protozoa and algae 2. Place a single cover slip (be sure that they are not stuck together) on top. Prepare a smear and simple stain of the material between your teeth. Streptococcus. Use a bought smear that has already been stained. using different lenses. Start with brightfield. 1. The objective here is to become familiar with your microscope: It is NOT important to identify protozoa or algae in the pond water. Practice with the microscope. Much of the algae will be there also. etc. Focus on the sample using 10X. then go to 40X (NOT 100X). MATERIALS NEEDED: pond water microscope slides cover slips oil dropper prepared slides of a bacterium (Bacillus. Begin looking at the specimen using brightfield microscopy (condenser setting on 0). Any slide that you use for a wet mount is returned to your slide box. 7. Spread the suspension over the glass so that it forms a thin layer.Page 12 OBJECTIVES: Prepare wet mounts using live samples. E. remove some solid material between your back wisdom teeth. coli. after washing with tap water. changing condensers settings. 1. then switch over to darkfield and then phase-contrast. 4. and mix it into a drop of water so that you have a suspension spread over the middle third of the microscope slide. 6. Place the drop on a microscope slide. NO COVER SLIP USED! . Take a sterile toothpick. Remove the cover slip and discard it in the red SHARPIES container on the bench.

Therefore. end up. o Place the slide. most of it too small to see. Focus on the sample using the 10X lens (Be SURE that you are on brightfield microscopy): you should see masses of purple material. 6. cleansing each side of the slide. 7. Ready to go to 100X now? Be sure to read the directions in the MICROSCOPY exercise so that you know how to move to the 100X objective lens using oil. The heat-fixing will coagulate some of the protein material and cause the suspension to adhere to the glass slide. You still use oil on them with the 100X oil-immersion lens. The procedure for cleaning smears is simple. Flood the smear with crystal violet: let sit for 1 minute. Use your finger to spread the paste on the slide. Blot the smear slide with your bibulous paper pad. Be sure to REMOVE any oil before replacing them on the trays. chains? Any slide that you use for smears is returned to your slide box. you have heat-fixed TOO MUCH. and place the slide on the wire mesh. Notice the arrangements of the bacteria---pairs. Heat-fix the dry smear by running the slide quickly through the flame a few times. depending on whether the dye is in a dropper bottle or in a jar: o Place the slide on the wire over the stain tray. 9. it will not wash off during the various steps of staining. Identify the various shapes and arrangements of bacteria in your mouth. Wash the slide WELL with distilled water. Make a paste with Bon Ami cleanser powder (located in shaker bottles at each sink) and water. cover slips are on them. into the jar of crystal violet for 1 minute. 3. Be SURE that the crystal violet dye is full in the jar (there are stock solution bottles in the cabinets to fill them). Let the slide air-dry—totally. Staining the smear can be done 2 ways. If 4. 8. your fingers get hot. to be cleaned and used again. Look at a bought prepared bacterial smear.Page 13 2. Most of them will be bacillus-shaped or coccus-shaped. BE SURE TO WIPE OIL OFF OF THE 100x LENS WITH LENS PAPER---not paper towel. clusters. but it would not be uncommon to see some spirilla. Since these are bought stained smears. not kim-wipes! WASH YOUR HANDS AFTER WORKING WITH MICROORGANISMS AND STAINS! . This step facilitates the fixing of the smear to the slide. 5. not bilulous paper.

Do you think that all basic dyes would have the same binding affinity for different bacteria? 3.Page 14 LABORATORY REPORT SHEET Draw your results seen in the microscope (using 100X lens) from the mouth from the bought prepared smear QUESTIONS: 1. Draw a bacillus-shaped bacterium. What would happen if you forgot to heat-fix the smear? 5. 4. What is a simple stain? 2. Why stain bacteria rather than looking at wet mounts of them? .

viral encephalitis o flea (Pulex) – plague caused by Yersinia pestis These ectoparasite vectors are in the Kingdom Animalia. they are called ectoparasites. are vectors for infectious diseases. (Pithirus) pubic louse– STD – the “crabs” o itch mite (Sarcoptes) – sarcoptic mange –STD . Ticks and mites are in the Class Arachnida (relative of spiders).. Examples are: o Hard body ticks (Ixodes) – Lyme disease caused by bacterium Borrelia burgdorferi (Dermacentor) – tularemia caused by bacterium Francisella tularensis. numbers of leg pairs. while the others are in the Class Insecta. generally in 2 classes---Insecta and Arachnida.“scabies” (not a microbial infection) o mosquito (Anopheles) – malaria caused by various species of the protozoan Plasmodium. Dept.ECTOPARASITES Page 15 Various arthropods. and presence of antenna.au/photos/insect%20photos. main body parts. (Aedes) – dengue (viral) fever.htm OBJECTIVES: Learn about various vectors of infectious diseases. Rocky Mountain spotted fever caused by bacterium Rickettsia rickettsii o Soft body tick (Ornithodoros) – Relapsing fever caused by bacterium Borrelia recurrentis o body louse (Pediculus) – typhus caused by various Rickettsia species. Univ. of Medical Entomology.edu.usyd. MATERIALS NEEDED: prepared slides of ectoparasites flea louse mosquito head tick (hard bodied and soft bodied) fly head THE PROCEDURES: View various prepared slides using the low power of stereoscopic microscopes. mainly from the Insect class. . of Sydney http://medent. Differences between the Insects and Arachnids include presence of wings. When living on or interacting with the surface of the body.

Why is malaria not common in the United States? . How does the body plan of Insects differ from Arachnids based on major divisions? 4. How many leg pairs does the Class Arachnid have compared to the class Insecta? 3.Page 16 LABORATORY REPORT SHEET QUESTIONS: 1. Record the views of the various parasites (on low power magnification) flea louse bedbug tick mosquito 2.

Platyhelminthes are flatworms. FEATURES OF HELMINTHIC GROUPS: trematodes (flukes) – flat. and one might wonder why they are covered in microbiology.HELMINTHS Page 17 There are many worms in the kingdom Animalia. the worms are transmissible diseases. you will definitely have to learn the eggs later. via insects. Mainly. . The life cycle of a worm can be very complex. Second. but we are looking at pathogenic representatives in 2 phyla (a major group within a kingdom is a phylum)---Platyhelminthes and Nematoda. segmented. separate sexes Low power 10X or even scanning power (using the stereoscopic dissecting microscopes) should be sufficient for these large organisms. The Nematodes are roundworms. probably with lots of eggs.gov/dpdx/ OBJECTIVES: Differentiate between flatworms and roundworms. divided into the cestode (tapeworms) class and the trematode (flukes) class. hermaphroditic nematodes (roundworms) – unsegmented. separate sexes cestodes (tapeworms) – flat. Helminths are multicellular. food. and are a bit more evolved than the platyhelminthes. with multiple hosts for different stages of the worm. They come in separate sexes. unsegmented.cdc. soil---similar to bacterial and viral infectious diseases. Recognize common features of each worm. On the other hand. The adult worms are macroscopic.dpd. leaf-shaped. if you are planning a career in clinical lab sciences (medical technology). water. First. Identify some common worms. you will see genital organs inside of the adult worms under the microscope. The egg slides are set up as demos only: You do not need to be able to identify the worms by their eggs. Differentiate between flukes and tapeworms. Excellent Resource on Parasites at Centers for Disease Control! Laboratory Identification of Parasites of Public Health Concern http://www. diagnosis of helminthic diseases relies on the microscopic identification of the eggs or larvae.

Page 18 MATERIALS NEEDED: prepared slides of the adult: (use stereoscopic microscopes for scanning power and 10X low power on your regular microscopes)
• • • • • • •

Taenia Schistosoma Fasciola Enterobius Trichinella Ancylostoma or Necator Dirofilaria (heartworm in dogs) larvae in blood or Wuchereria (filariasis in humans)

preserved specimens of various worms demo microscopes with eggs of adults (just for show, NOT for identification) THE PROCEDURES: Major features of each organism are listed to the right. THE WORM: class and genus key features to notice

PLATYHELMINTHES
TREMATODES Schistosoma adult DEMO: egg Fasciola hepatica adult (sheep liver fluke) DEMO: egg CESTODES Taenia solis or T. saginata adults DEMO: egg Scolex head with suckers and/or hooks, proglottid segments (with reproductive organs)

adult male and female copulating, suckers eggs within body, suckers

NEMATODES
Enterobius vermiformis adult (pinworm) DEMO: egg Necator or Ancylostoma adult (hookworm) DEMO: egg Trichinella spiralis larvae in muscle Dirofilaria immitis filarial larva in blood smear eggs in body cutting teeth, tail of male vs. female encystment

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LABORATORY REPORT SHEET
QUESTIONS: 1. Draw the organisms. Taenia Schistosoma Necator/Ancylosoma

Fasciola

Enterobius

Trichinella

2. Is Schistosoma a trematode, cestode or a nematode? 3. Which group of helminths has a scolex? 4. The meaning of hermaphroditic? 5. Why are Schistosomes called blood flukes? 6. Which group of parasites has flat and segmented body? 7. Give one way in which roundworm Dirofilaria or Wuchereria differ from the other worms.

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then dividing continuously. in air.52% agar for the solidified media. It is estimated that only 3% of bacteria are pathogenic for man and animals. Commonly. Most of them are part of a commensualistic symbiotic relationship (they benefit. Generally. the presence of bacteria will produce a turbidity (sterile broth medium will be clear). in water. In fact. Label the bottom of your agar plates with your name. they are contributors to the environment. The difference is the presence or absence of the complex polysaccharide called agar agar. piled on top of each other in a discrete area of the plate. Your table will sample the environment in 2 ways: a. containing vast numbers of bacterial species. etc. a solidifying agent purified from red algae. Bacteria are both metabolically diverse as well as structurally diverse. it is added as a 1. in your large intestine alone you harbor more bacterial cells than the total number of human cells in your body. in soil. MATERIALS NEEDED: 3 TSA (trypticase soy agar) agar plates per table (2 for environment. Become familiar with the use of various forms of media. 2. will produce colonies on the agar surface. Two forms of bacteriological media will be used to culture the microorganisms---agar medium and broth medium. You will undoubtedly see mixed cultures from most of your specimens---different colony sizes. date. and location of specimen. lab section. Bacteria are found in a wide variety of environments---in or on animals and plants. Your body is actually its own ecosystem. These indigenous bacteria. Agar agar is not a nutrient: it cannot be utilized by the organisms. ISOLATION OF BACTERIA FROM THE ENVIRONMENT 1. colors. OBJECTIVES: Compare the growth of bacteria from different environments. shapes. giving a solid surface for the bacteria to grow on. air sample b. you are neither harmed nor do you benefit) or a mutualistic (both partners gain) symbiotic relationship. surface of an object (not on the body) 3. decaying nutrients and recycling the minerals (for use by plants and other organisms). 1 for body) 1 TSB (trypticase soy broth) per table (tables doing lips and sneeze do not use TSB) THE PROCEDURES: Each table will sample 4 habitats---2 from the body and 2 from the environment. are part of your normal flora. For surfaces of objects: .THE UBIQUITY OF BACTERIA Page 21 Bacteria far outnumber all other life forms on the planet. Identify the ways that bacteria grow on and in bacteriological media. living in and on your body. Each colony started with one mother cell deposited on the agar surface. or on rock. On an agar plate clusters of daughter bacterial cells. In a broth culture.

Do the same with a tube of TSB. lips and sneeze plates done on agar plates only. back teeth swabbed Lips lightly pressed against agar medium Sneeze onto plate ISOLATION OF BACTERIA FROM THE HUMAN BODY 1. Roll the swab around on the assigned environmental area.9% NaCl) and squeeze the swab against the glass wall of the tube to reduce excess fluid. Incubate the agar plate and broth tube at room temperature. Label the bottom of your agar plate with your name. 3. c.Page 22 a. dip it into a tube of sterile saline (0. 6. Rub the swab on an agar plate. 4. no TSB. 25 degrees Celsius. For air specimens. Taking a sterile cotton swab from the package. using the zig-zag procedure shown below. and location of specimen. expose your TSA plate to your assigned area by uncovering the agar cover. ENVIRONMENTAL SPECIMENS Air in lab for 30 minutes Air in toilet stall (at base of toilet) for 30 minutes Air outside of building for 30 minutes Toilet bowl inside swabbed Toilet bowl outside swabbed Various coins placed on medium Paper currency pressed onto agar medium Hair shaken over plate Doorknob swabbed Cell phone speaker swabbed Cell phone earpiece swabbed BODY SPECIMENS Palm of hand swabbed Sole of foot swabbed Inside elbow swabbed Cheek swabbed Between fingers swabbed Armpit swabbed Inside ear swabbed Inside nose swabbed Inside mouth. using a new swab and the same area of the body on the opposite side. b. 25 degrees Celsius. being sure to write on masking tape to label the tube. . 5. Place the swab into a tube of sterile TSB and cap the tube. Rub the swab on an agar plate. Taking a sterile cotton swab from the package. date. 7. dip it into a tube of sterile saline (0. lab section. Roll the swab around on the assigned area of your body. using the zig-zag procedure shown below.9% NaCl) and squeeze the swab against the glass wall of the tube to reduce excess fluid. 5. Repeat steps 2 and 3. Incubate the agar plate at room temperature. 4. 2.

2. Try to differentiate different species of bacteria by colony shapes. and pigment.Page 23 DATA COLLECTION: BACTERIAL COUNTS FOR AGAR MEDIA 0 = no growth +1 growth = 10 bacterial colonies or less +2 growth = 10-100 colonies +3 growth = >100 colonies 1. 2. 4. along with the source of the specimen. Check for the number of bacterial colonies. 3. Try to differentiate different species of bacteria by colony shapes. Check for the number of bacterial colonies. GROWTH IN BROTH MEDIA 0 = clear +1 = light turbidity +2 = medium turbidity +3 = very turbid. back teeth Lips lightly pressed against medium Sneeze onto plate . cannot see through broth 1. Record the amounts of growth in broth cultures and agar plate cultures. Amount of growth Different species # ENVIRONMENTAL SPECIMENS Air in lab for 30 minutes Air in toilet stall (at base of toilet) for 30 minutes Air outside of building for 30 minutes Toilet bowl inside swabbed Toilet bowl outside swabbed Various coins placed on medium Paper currency pressed onto agar medium Hair shaken over plate Doorknob Cell phone speaker swabbed BODY SPECIMENS Palm of hand Sole of foot Inside elbow Cheek Between fingers Armpit Inside ear Inside nose Inside mouth. The entire class’s data will be listed on the board. size. and pigment. size.

Can you determine the number of different bacterial species in a broth culture? Explain. which one gave the great variation in bacterial species? . Which of the body areas had the highest counts? 4. 2.Page 24 LABORATORY REPORT SHEET QUESTIONS: 1. Did any habitats result in no growth? 5. Which of the environmental habitats had the highest counts? 3. Of the specimens taken from the body.

The lichens are actually mutualistic. absorbing organic material from their environment. Identify key representatives of each class. although they more commonly reproduce by asexual budding. Aspergillus) http://www. The hyphal filaments are haploid (1N). In addition. The majority consists of microscopic filaments called hyphae. to name just a few. Their cell walls contain chitin a polymer of the sugar glucosamine.doctorfungus. i.e. The classes of fungi are based mainly on the type of sexual spore that is produced. having names such as sporangium. heterotrophic. Fungus UC Berkeley's Introduction to Fungi OBJECTIVES: Identify the various classes of fungi and major features among them. Ascomycota. Saccharomyces. and so on. sucrose. fungi make up part of the composite organisms called lichens. and Candida albicans prepared slides of dermatophytes (Microsporum. Epidermophyton) fresh cultures of fungi on agar plates (Rhizopus. asexual spores are the more common spores. The sexual spores are produced by meiosis. There are quite a few classes of the kingdom Fungi---Chydridiomycota. you will identify representatives from 3 categories of fungi: • Basidiomycetes (representative: mushrooms) • Ascomycetes(representative: Penicillium.html . budding. On the other hand. glucose) prepared slides of Rhizopus prepared slides of Penicillium and/or Aspergillus. ascospore. There are various reproductive modes for production of asexual spores---fragmentation. basidiospore. Penicillium. nonphotosynthetic organisms in a separate kingdom of the same name. In this lab. and basidium. symbiotic relationships between fungi and photosynthetic algae or photosynthetic cyanobacteria. They live either as parasites or as saprophytes.berkeley. Trichophyton. Fruiting structures arise from the mycelium. fission. Zygomycota. various dermatophytes) • Zygomycetes(representatives: Rhizopus) Great Resources! Dr. These fruiting structures can contain sexual spores or asexual spores. Basidiomycota. ascus. zygospore. and are often contained within a structure.due to a lack of currently defined sexual spores. and the network of filaments is the mycelium.org/ http://www. and Deuteromycota. MATERIALS NEEDED: culture of Saccharomyces cerevesiae phenol red sugar broths with durham tubes (lactose. The Deuteromycota group contains the unclassified fungi that mycologists don't really know where to put. their function being dispersal so that the fungus can disseminate itself throughout the environment. Even yeasts produce sexual spores.ucmp.edu/fungi/fungisy.FUNGI Page 25 Fungi are eukaryotic.

conidia fruiting structures.Page 26 THE PROCEDURES: 1. • Look at prepared smears of mixed yeasts (Saccharomyces and Candida) • Inoculate the three sugar broths (lactose. and then break off. . Rhizopus prepared slides If 2 different strains (called + and – strains) are placed together on a culture medium (or in nature). sucrose and dextrose) using the culture of S. 2. Penicillium and Aspergillus On 10X and 40X. DEMOS only! Dermatophyte genera: Microsporum. Use phase-contrast or brightfield microscopy. Use brightfield microscopy. small daughter cells arising from the mother cell. After incubation observe the tubes for fermentation of the sugars (a change in color of the phenol red indicator from red to yellow) and also note if carbon dioxide gas was produced by looking for bubbles in the inverted Durham tube. and the asexual conidiospores. • On 10X and 40X. • Differentiate between the sexual zygospores and the sporangiospores on the slides. 4. identify various dermatophytes’ fruiting structures. sporangia. Incubate at room temperature until the next laboratory session. Trichophyton. the hypha will grow towards each other and conjugation will occur. 3. identify hyphae. Saccharomyces cerevesiae Yeast reproduce asexually by budding. identify hyphae. Epdermophyton On 10X and 40X. cerevisiae provided. • Make a smear of the yeast and simple stain with crystal violet. • Make a wet mount of the culture (SMALL inoculum) in a drop of lactophenol cotton blue (10X and 40X). and sporangiospores. They will stay attached until disturbed. This produces a sexual spore called a zygospore—a diploid sexual spore.

By what major criterion are the fungi subcategorized into classes? 6. 6. Record the shapes and features of the various fungi.Page 27 LABORATORY REPORT SHEET QUESTIONS: 1. 5. Name a couple of ways in which the molds and yeasts differ. The mushrooms are in what class of fungi? 3. Saccharomyces Penicillium Aspergillus Candida Rhizopus asexual spores Rhizopus sexual spores 2. 3. Which one of those fungi is commonly known as brewer’s or baker’s yeast? . Are the conidiospores of Penicllium and Asperillus arranged differently ? 8. Differentiate hypha and mycelium 7. Is a zygospore a sexual spore OR an asexual spore? WHY? 4.

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In addition. dormant structure called a cyst. Euglena) • Ciliates (representative: Paramecium) • Apicomplexa (representative: Plasmodium) Many protozoa are found in the gut of warm-blood animals and cold-blooded animals. They do not photosynthesize. The flagellates have flagella or an undulating membrane for motility. even hosts. This means that they use chemicals for energy production and they get their carbon from the same compounds.magnet. called the apical complex (used in the takeover of the host cell). The ciliates have cilia. EXTERNAL LINKS: Pond Water Critters – http://microscope-microscope. called a trophozoite. both of which may be found in the feces. there are only 4 groups of protozoa that will be covered here: these groups are separated by motility and cell structure.fsu. in addition to the cyst stage.g. This last class has most of the human and animal pathogens in it. Many of the protozoa form a resistant. some of which may have tests or 'shells' that surround their cytoplasm.htm movies of protozoa at Molecular Expressions http://micro. .org/applications/pond-critters/pond-critters.html OBJECTIVES: Identify common species of protozoa. sugar. Parasitic protozoa are identified by the active feeding stage.PROTOZOA Page 29 The protozoa are contained within the kingdom Protista along with the unicellular algae. You will likely see some freshwater amebas in the pond water. there are quite a few protozoans that live in blood. for example. For our purposes. The Apicomplexa have a unique arrangement of microtubules. in the cell. Differentiate among the major classes of protozoa. e. as well as in insects such as termites and cockroaches. Amebas move by cytoplasmic streaming.edu/moviegallery/pondscum. rather being chemoheterotrophic like animals. motility structure. there are some pigmented protozoa and there are even a few that seem to be crossover organisms. being claimed by the botanists because of their photosynthetic ability. However. having no motility structure. • Amebas (representative: Ameba proteus) • Flagellates (representative: Trypanosoma. The classes of protozoa are categorized by a variety of factors: cell architecture. You will see some of these examples in lab. the flagellate Euglena will photosynthesize in light (it contains chlorophyll) or will switch over to regular aerobic respiration (chemoheterotrophism) without light. Identify different types of motility.

Observation of prepared. Do NOT stir the specimen: you will get fewer that way.Page 30 MATERIALS NEEDED: prepared slides: • Trypanosoma • Plasmodium fresh specimens: • Ameba • Paramecium • pond water cover slips pipets for specimens THE PROCEDURES: 1. 2. change over to darkfield and phasecontrast for even better viewing. . go down to the bottom of the container or in the algae and dirt to get your specimen to make a wet mount slide. and so have to be viewed with 100X magnification. bought slides of blood parasites Trypanosoma Plasmodium Use brightfield microscopy. • Start with the 10X and go to 40X. • Place the drop on a microscope slide and cover with a cover slip. • Once you have found your objects on brightfield. Oil-immersion will magnify too much for most pond water protozoa. Begin on brightfield microscopy. Plasmodium will be difficult since the parasite will be inside of the RBCs. Start with the 10X objective lens. ending up on 100X Trypanosoma will be easy to see: it is far larger than the red blood cells. Be sure that you have one cover slip only to place on top of the slide. However. Preparation of wet mounts using live protozoa Paramecium Ameba pond water • Using the pipet provided. These are small protozoa.

Paramecium Ameba Trypanosoma Plasmodium 2.Page 31 LABORATORY REPORT SHEET QUESTIONS: 1. Which of the organisms seen in lab are intracellular parasites? . By what major criterion are the protozoa subcategorized into classes? 4. In which class does Plasmodium reside? 6. What organisms are in the kingdom Protista? 7. Draw the organisms seen. What motility structure does Trypanosoma use? 5. Where is the malarial parasite Plasmodium located---in the RBC or in the plasma outside of the RBC? 3.

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) THE PROCEDURES: 1. First. This simple stain procedure has already been done. size) of the bacterium. This basically involves 3 steps----transferring a liquid suspension of the bacterium on the slide. This will help you later in locating the smear. Bacillus. the staining procedure begins. using material from your own mouth. sometimes a problem when moving the slide back and forth looking for your bacteria. works well to remove the smear and the dye from the glass. MATERIALS NEEDED: TSB culture of Staphylococcus epidermidis TSA plate of E. Use the microscope to identify features (shape.SIMPLE STAINING & SMEAR PREPARATION Page 33 In order to stain the bacterial specimen for microscopy one must first prepare the smear on the slide. One needs to be careful about thick smears when taking the specimen from an agar medium. drying the smear. in a previous lab---PREPARATION OF SPECIMENS. Refer back to the discussion and procedure in that lab exercise. and then heating slightly to firmly attach the smear to the slide. Prepare a simple stain of the smear. Once this is done. We recycle our microscope slides and the smears have to be removed from the glass. bought slides of stained bacteria (mixed shapes. make sure that you have a few clean slides. arrangement. CAUTION: Do NOT make your smear suspensions too thick: The dye will not penetrate well. The wax pencil is better than a marker because it will not wash off easily from the glass. . coli dye rack for staining dye kit with reagents clean microscope slides wax pencil blotting paper immersion oil prepared. OBJECTIVES: Prepare a smear from a bacterial specimen. and there will be far too many bacterial cells to see individual shapes and arrangements. etc. a cleansing powder similar to Ajax or Comet (but not as abrasive on the glass). RECOMMENDATION: You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. Bon Ami.

Heat-fix the dry smear by running the slide quickly through the flame a few times. Return prepared slides to the trays on the bench. 9. 15. 13. . Wash the slide WELL with distilled water. 11. o Dry the slide. 10. You will see different shapes and arrangements. You will make smears using 2 different types of cultures. remove a small inoculum (use only a small amount of large colonies) with a loop and suspend it in a small drop of distilled water.Page 34 At the sink. The heat-fixing will coagulate some of the protein material and cause the suspension to adhere to the glass slide. o Shaking the culture first. and then wash WELL with tap water. pick up the bottle of Bon Ami. 6. Focus on the sample using the 10X lens (Be SURE that you are on brightfield microscopy). into the jar of dye for 1 minute. Refer to the lab exercise on MICROSCOPY for help with the microscope. 3. NO COVER SLIP USED! 5. Flood the smear with the dye: let sit for 1 minute. Staining the smear can be done 2 ways. o From the agar plate culture. end up. Focus on the smear using the 100X oil immersion lens. depending on whether the dye is in a dropper bottle or in a jar: o Place the slide on the wire over the stain tray. coli. 12. Look at the prepared slides of various bacteria. aseptically transfer a drop of the TSB culture using an inoculating loop to a clean slide. Return dirty slides to your slide box. Add a couple of drops of tap water and make a thick suspension (consistency of toothpaste). Mix the culture well into the water drop. This step facilitates the fixing of the smear to the slide. Spread the smear suspensions over the glass slide so that it forms a thin layer and it is the size of nickel or larger. coli and crystal violet Staphylococcus and safrinin 8. Be SURE that the dye is full in the jar (there are stock solution bottles in the cabinets to fill them). o 2. o Use your finger to spread the Bon Ami suspension over each side of the slide. o Label the 2 slides with the names of your bacteria. 7. a broth and an agar culture. Be sure to read the directions in the MICROSCOPY exercise so that you know how to move to the 100X objective lens using oil. you have heat-fixed TOO MUCH. and place the slide on the wire mesh. 4. shaking some on top of the smear on the slide. 14. A different dye will be used for each bacterium--E. Identify the various shapes and arrangements of bacteria using the fields below. o Place the slide. Let the slide air-dry—totally. If your fingers get hot. Blot the smear slide with your bibulous paper pad. Label 2 slides---Staph and E.

Why heat-fix the smear? 6. What names are given to the shapes seen in the 2 bacteria used in lab? 5. Are all bacteria the same size? 4. Since everything on the slide will be the same color in simple staining.Page 36 LABORATORY REPORT SHEET QUESTIONS: 1. Record your results of staining. what you possibly tell about the organisms? 3. E. What is the disadvantage of having a really thick smear when staining? . Why are basic dyes more useful than acidic dyes when staining bacteria? 7. coli Staphylococcus 2.

the type of organism (acid-fast bacteria and spores do not stain well). the cultures should be 18-48 hours old). the time of decolorization. Differential stains use 2 or more dyes. The most common reasons for false gram reactions? • Some bacterial species tend towards gram variable. Gram was actually using dyes on human cells. originally developed in 1884 by Christian Gram. is probably the most important procedure in all of microbiology. thickness of the smear. Although there is a standard routine and set reagents used in this stain. During the crystal violet-iodine step. Before staining. the outer lipopolysaccharide layer of the wall is dissolved by the decolorizer agents. The many variables that can affect this stain are age of the culture. CAUTIONS: . too long a time. Gram positive bacteria retain the crystal violet even through the decolorizor step: gram negative bacteria do not retain the crystal violet. are decolorized. • Over decolorizing the smear. as opposed to the simple stain done recently. and found that bacteria preferentially bind some dyes. the crystal violet is leached out of the wall. and will show both colors although most often gram +. The acetone-alcohol actually causes the peptidoglycan molecules (arranged in a latticework) to shrink. Because of the 2 dyes used in the procedure---crystal violet and safrinin---as well as the decolorizer acetone-alcohol. producing different colored cells depending on their chemical and physical properties. thereby holding the crystal violet-iodine even tighter. amount of decolorizer used. and then pick up the safrinin dye. The Gram stain is a differential stain. each person has to find a particular method that works best for them. It has to be one of the most repeated procedures done in any lab. and the general care of the stainer. the specimen must be mounted and fixed on the slides. In the gram – cell. as previously done in the simple staining technique. and because the peptidoglycan layer is so thin in that group of bacteria. the bound molecules within the peptidoglycan of the gram + cell wall are held tightly. Both gram + and – bind to the crystal violet: the key step to their differentiation is the decolorization. • Using old cultures (preferably. Take a look at the accompanying diagram of the stain procedure and its effects on the bacterial color. bacteria will fall into 2 groups based on their gram reactivity.THE GRAM STAIN Page 37 The gram stain.

. gram stained slides of bacteria of different shapes and sizes of bacteria to look at. Make sure that you have all of your materials---dye kits. MATERIALS NEEDED: unknown bacteria on various culture media Gram negative control: E. Let the slide air-dry totally before proceeding on with the procedure. preferably a quarter. o If taken from a broth. Differentiate among various shapes. making smears of the 2 control bacteria along with the unknown bacterium. use 1-2 loopfuls of the broth solution. suspend the inoculum in a drop of water on the slide and mix it well. 1. slides. once you have started the stain procedure. 5. Refer back to the exercise SIMPLE STAINING to read about cleaning microscope slides and how to make smears. cultures. o If taken from a solid agar medium (plate or slant). o Spread the suspension on the slide so that it covers an area at least the size of a nickel. looking for materials. o Place a piece of tape on the side of the smear so you know which way is UP. 4. 2.Page 38 o Make sure that your smear is not too thick: otherwise. This will give you gram + and gram . rack. slant. bought slides of various gram stained bacteria dye kit clean microscope slides dye stain rack and container immersion oil blotting paper THE PROCEDURE: done individually We have cultures of E. o Flood the smear with decolorizing agent evenly over the smear. water--available. coli on TSA slants or plates Gram positive control: Bacillus subtilus in TSB prepared. and gram reactions of bacteria. either the dyes cannot penetrate or the cells will not be decolorized adequately. o Be sure that you properly time the decolorization step. o Practice will give you consistent results. There are also prepared. o You might think about marking the smear area with a surrounding wax pencil mark so that you can find the smear under the microscope easily. or plate. arrangements. coli and Bacillus for your table to gram stain also. Heat-fix the slide by holding the slide at one end with your fingers and quickly moving it back and forth a few times over the flame. sizes. OBJECTIVES: Become proficient at performing the gram stain consistently and accurately. 3. It is helpful to divide the slide into 3 sections. You do not want to be scrambling.controls to check your procedure against. Make the bacterial smear from broth.

leaving in for 1 minute. o Blot dry with bibulous paper. 7. . leaving in for 1 minute. STAIN procedure o Place slide into container of crystal violet. Focus on smear using low power lens. Wash WELL with water.Page 39 6. Be sure that you have a drop of oil on the slide before rotating your 100X objective lens into place. Wash WELL with water. ending up on 100X oil immersion. Wash WELL with water. o Place slide into container of safrinin. o Flood acetone-alcohol quickly on the slide. o Place slide into container of Gram's iodine. and wash off within 5-10 seconds (from beginning of decolorizer added). leaving in for 1 minute. Wash WELL with water.

If a gram stain gives you some valuable information on features of the bacterium. Would it be useful to perform a gram stain on a mixed culture? Why? 7. coli when gram stained? Name the dye that gives it this color. coli? 3. what would be the benefit of ever performing a simple stain? . What color is E. List at least 3 differences between gram positive and gram negative bacteria. 2. What are the gram reaction. 6. Critiquing your gram stain technique: o Are the cells well distributed on the slide? o Are the cells stained uniformly and is the gram reaction correct? o Is the arrangement of the bacterium consistent across the various fields of vision? 2. and arrangement of Bacillus? What are the gram reaction.Page 40 LABORATORY REPORT SHEET QUESTIONS: 1. To what cell structure do the 2 dyes bind? 5. shape. 4. and arrangement of E. shape.

if washed well with water. air-dry. Some spore-forming species produce spores only under adverse environmental condition.THE ENDOSPORE STAIN Page 41 Endospore production is a very important characteristic of some bacteria. Spores will be a light green: Vegetative cell walls will pick up the counterstain safranin. In fact. . extreme heat. and heat-fix. tetanus. Endospores are not for reproduction: One spore forms inside of the vegetative cell. desiccation. one vegetative cell will be produced. MATERIALS NEEDED: dye kit dye stain rack hot plate paper towel (cut the size of the slide) Bacillus culture on an TSA plate THE PROCEDURES: 1. chemicals. heat. The primary dye malachite green is a relatively weakly binding dye to the cell wall and spore wall. allowing them to resist adverse environmental conditions such as desiccation. 2. The identification of spores is also very important for the clinical microbiologist who is analyzing a patient's body fluid or tissue since there are not that many spore-forming genera. chemical exposure. while a few species easily produce spores without much prodding. Identify spores on a bacterial smear. That is why there does not need to be a decolorizer in this stain: it is based on the binding of the malachite green and the permeability of the spore vs. etc. making it increasingly resistant to the staining dyes. Prepare the bacterial smear of Bacillus. however not from the spore wall once the dye is locked in. When the spore germinates. Bacterial endospores are the most resistant structures of all living organisms. to name a few. This lab will employ the Schaeffer-Fulton Method: there are variations of the spore stain method. and so a gimmick—steaming---enhances the primary dye’s penetration. and anthrax. the dye comes right out of the cell wall. OBJECTIVES: Learn to perform the spore stain. As a spore forms inside of the vegetative cell. cell wall. gangrene. there are two major pathogenic spore-forming genera. the spore wall chemically changes and thicken. etc. This sporulation process changes the spore’s stainability. Bacillus and Clostridium. Put a beaker of water (a metal beaker) on the hot plate and boil until steam is coming up from the water. and they can live in this dormant dehydrated state for hundreds and hundreds of years (even some documented at thousands of years). Then turn the hot plate down so that the water is barely boiling. The stimulus for sporulation can vary—nutrient depletion. In fact. together causing a number of lethal diseases---botulism.

if there is one---terminal. If you mistakenly did this stain on an acid-fast Mycobacterium. 4. Wash really WELL with water and move the slide and wire rack from the boiling water to the regular stain tray to finish up the last step in the procedure. DO NOT let the dye dry on the towel. The towel will keep the dye from evaporating too quickly. 9. 7. Place the wire stain rack over the beaker which now has steam coming up from the boiled water. Cut a small piece of paper towel and place it on top of the smear on the slide. Why might that information be helpful to a microbiologist? 3. 6. Why does there not have to be a decolorizer in this stain? 5. 5. What is the purpose of the steam in this stain? 4. and leave for 5 minutes. Flood the smear with the primary dye. safrinin. Wash WELL with water. Can you find any spores still within the vegetative cell? If so. 2. LABORATORY REPORT SHEET QUESTIONS: 1. 8. what color do you think the cells would come out to be? . subterminal.Page 42 3. Remove and discard the small paper towel piece in the trash. and leave for 1 minute. thereby giving more contact time between the dye and the bacterial walls. Draw some spores along with some vegetative cells for comparison. Blot dry with bibulous paper. central. Keep the paper towel moist with the malachite green. notice where the spore is in the cell. Place the smear in the stain jar or flood the smear with the counterstain dye. malachite green.

caused by 2 different species of the genus Mycobacterium (commonly called AFB in clinical situations). Place the wire stain rack over the beaker which now has steam coming up from the water. and leave for 1 minute. . Put a beaker of water (metal beaker) on the hot plate and boil until steam is coming up from the water. air-dry. Remove the piece of paper and discard in the garbage. 8. Cut a small piece of paper towel and place it on top of the smear on the slide. Once in. You will make a mixed suspension of Mycobacterium and E. have a high concentration of mycolic acid. Differentiate between acid-fast and nonacid-fast bacteria. thereby giving more contact time between the dye and the bacterial walls. Add the decolorizer acid alcohol (1% HCl + ethanol) and decolorize for 15-20 seconds. in their walls. Keep the paper towel moist with the carbol fuschin. and heat-fix. 3. Flood the smear with the primary dye. Acid-fast bacteria are hot pink or fuchsia. MATERIALS NEEDED: cultures of Mycobacterium smegmatis and E.THE ACID-FAST STAIN Page 43 The ability of the bacteria to resist decolorization with ACID alcohol confers acid fastness to the bacterium. 2. it will not come out: But the acid alcohol decolorizer WILL take it out of the nonacid-fast wall since the primary dye does not bind strongly to the cell wall. The towel will keep the dye from evaporating too quickly. 10. coli dye kit + dye stain rack hot plate and beaker paper towel (cut the size of the slide) THE PROCEDURES: 1. OBJECTIVES: Learn to perform the acid-fast stain. steam is used as a gimmick to get the carbol fuschin primary dye to go into the wall. Nonacid-fast bacteria are light blue. and leave for 5 minutes. 6. a lipid. carbol fuschin. This differential stain is very important for diagnoses of leprosy and tuberculosis. DO NOT let the dye dry on the towel. coli on one slide. 5. Blot dry with bibulous paper. Wash WELL with water. methylene blue. Flood the smear or place the slide into a jar with the counterstain dye. Prepare the bacterial smear. Wash the slide really WELL. Then turn the hot plate down so that the water is barely boiling. 4. As in the spore stain. 9. of which there are very few---the major genus Mycobacterium. Rinse with water. The method used in this lab is the Ziehl-Neelsen method: (there are 2 different methods of AF staining). 7. Acid-fast bacteria.

coli when gram stained? Acid-fast or not acid-fast? . although gram +.Page 44 LABORATORY REPORT SHEET QUESTIONS: 1. What is chemically unique about the Mycobacterium genus that causes it to be acidfast? 2. coli when acid-fast stained? What was the color of E. How is this stain procedure similar to the spore stain procedure? 3. is never becomes a dark bluepurple but rather stays a light purple. If you gram stain Mycobacterium. Why? 4. What is the color of E.

OBJECTIVES: Identify capsules around bacteria. MATERIALS NEEDED: prepared slides of encapsulated bacteria THE PROCEDURES: These stains are bought and ready to use. you still use oil when on 100X magnification. Although they have cover slips. one acidic and one basic. It is nonionic so that the dyes that we commonly use will not bind to it. respectively. . Two dyes. The capsule gives added protection to the bacteria.THE CAPSULE STAIN Page 45 The capsule is a thick polysaccharide or polypeptide(or both) layer around the outside of the cell. are used to stain the background and the cell wall. Be sure to remove the oil with the lens paper. making it virtually impossible to be phagocytosed by white blood cells.

Page 46 LABORATORY REPORT SHEET QUESTIONS: 1. What is the function of the capsule? 3. What is phagocytosis? . Why does the capsule NOT take in any dye? 2. What does that mean? 4. The capsule is a type of negative stain.

• You always pick up your microorganisms as a set for your table. • Wash your hands with soap both BEFORE and AFTER lab. • Keep test tube caps and petri dish covers on media to reduce contamination (matters not whether it is sterile media or already cultured).5-2%) as a solidifying agent for isolation and purification. luxuriant growth. when you have a spill.TRANSFER OF BACTERIA USING ASEPTIC TECHNIQUE GENERAL GUIDELINES: Page 47 Safety • Wear a lab coat and have your goggles on! • ALWAYS disinfect the tables BEFORE and AFTER lab. • Do not dump ANY microbial suspension down the drain--only in the discard area. o Flood the area of the spill with disinfectant and leave on for 10 minutes before using paper towels to soak up. date. and. THE LAST PERSON ON THE TABLE CHECKS TO MAKE SURE THAT ALL 4 GAS JETS ARE OFF! • If there is a spill: o Tell your instructor about it. • Check for contamination in or on media. • A semi-solid has a reduced percentage of agar. Technique • ALWAYS use the proper aseptic technique when transferring cultures from one medium to another. • Place test tubes in racks when working at your table: never lay the tubes down--they leak. • Be sure to have nonessential materials (the ONLY essential thing is the lab handout and a notebook to write in) off of the table. • Use masking tape only to mark on the tubes: You can use tape or mark directly on the agar plates. • A broth medium has no agar: it is for fast. o Tubes in test tube rack discard o Agar plate in autoclave bag • REMOVE ALL labels from test tubes when discarding. • The gas should be turned all of the way on. and. Beginning the lab • Label all test tubes and petri plates with your name (initials). Finishing the lab • When using microbial cultures. Remove contaminated media and place in discard area. so that the level is parallel with the rubber tubing. and name of organism. use it. and then DISCARD properly. and discard them after use. your table #. therefore. take one of each organism for the table. in addition. exercise #. sharing them between the table members. There are 3 forms of media: • An agar medium contains agar (1. By now you know what media is: it is the nutrient-rich material that provides food to the microbes. • Be sure to put any unused media tubes BACK into the racks if unused: Replace unused agar plates back into bags if not used.. has qualities of both agar and broth. .

WHY? • It reduces bacterial contamination since the bacteria. Do the same with any tube media that you pick up. If contaminated. In this exercise you will learn how to subculture bacteria. or plate If you see water running on the agar plate. Each colony represents a different clone of cells. Identify different ways by which bacteria grow in culture—in agar deeps. each originating as a single mother cell. in broths. using a variety of culture media as your inocula sources and as your new culture media. Different species of bacteria will be used so that you can become familiar with different growth patterns.Page 48 In addition to nutrients and growth factors. MATERIALS NEEDED: set of cultures for the table: a TSA slant culture of Bacillus subtilus a TSB culture of Staph epidermidis a TSA plate of E. and perhaps agar. • It reduces the possibility of water condensation that may be on the lid dropping onto the agar. causing fluid to run across the agar medium. Eliminate potential contamination of bacterial cultures by using aseptic technique. discard the plate in the autoclave bag. on agar plates. even if they get into the plate in between the lid and bottom. Practice hand coordination required in good transfer techniques. pH buffers. You will also have a mixed culture with 2 species in order to learn how to best separate and isolate bacterial species. OBJECTIVES: Subculture bacteria in/on sterile media of various forms. All agar plates are incubated UPSIDE DOWN (exceptions will be pointed out occasionally). on agar slants. and pH indicators that allow biochemical reactions to be identified. which can then be isolated and grown as pure cultures on fresh media. there are additives such as NaCl salt. coli sterile media: (per person) 1 TSB 1 TSA slant 1 TSA plate 1 TSA agar deep THE PROCEDURES: BE SURE TO READ OVER THE STEP-BY-STEP DESCRIPTION OF TRANSFER TECHNIQUE BELOW BEFORE PERFORMING THE TRANSFERS. ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so. • Get another agar plate. you can do 2 things: • Place the agar plate upside down in the 37C incubator with the top cracked. . would have to go UP to get to the agar. Streak plates are a great technique for separating mixed cultures into visibly separate species. discard the tube.

and then going into the broth with the loop. --FROM AN AGAR PLATE CULTURE: The plate cultures have isolated colonies of bacteria growing on . • Use an inoculating needle for agar deeps and an inoculating loop for the agar plate and the broths. remove both tube caps with your little finger. Incubate all newly-made cultures at 25 or 37 degrees C. Subculture a plate culture of E. remove the inoculum and QUICKLY place the inoculum into the new medium tube. 7. Have both the culture that you are taking the inoculum from and the new. 2. 2nd session: 1. Be sure that you have all inoculating equipment handy. Heat the inoculating wire of the loop or needle again before placing on the table. ASEPTIC TECHNIQUE: 1. Check each culture for the presence of bacterial growth. 4. coli onto a TSA slant and onto a TSA plate. and in deeps. 6. Use the interpretation section at the end to determine the bacterium’s growth characteristics on a slant. and be sure that the ENTIRE wire is sterilized. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and replace the caps. Run the tops of the tubes through the heat to create an updraft (taking air contaminants AWAY from the tube entrance).Page 49 THE TRANSFERS: (performed individually) 1ST session: 1. Subculture a slant culture of Bacillus subtilus into TSB. Pick up both tubes in the hand not using the inoculation instrument. • Be sure to COOL your inoculation instrument a few seconds before picking the inoculum. TAKING THE INOCULUM --FROM A BROTH CULTURE: The inoculum is obtained by first shaking the culture a bit. 5. wherever your instructor directs you. Holding the tubes at a 45 degree angle. Incubate the plates and tubes on the 25 degree C shelf. Check the agar plate culture for growth. like a scraping motion. 4. 2. 8. 9. 2. 3. --FROM AN AGAR SLANT CULTURE: The inoculum is picked off of the top of the slant. 3. Be sure that the new medium is already labeled so you do not confuse the various cultures. Heat the inoculating wire of the loop or needle until red-hot. If you hear a sizzle as it touches the medium. it was TOO HOT. in broth. Keeping the sterile inoculation instrument in your hand. A film of broth culture can be seen across the loop as you remove it from the tube. You are now ready to pick the inoculum from the bacterial culture. sterile medium in front of you. (room temperature) Look at the section below in INTERPRETION to read your tube results. Subculture a broth culture of Staph epidermidis into a TSA deep.

you will learn how to make a streak plate but for right now just inoculate the agar plate with a zig-zag motion from top to bottom of the plate. check the growth patterns of all tubes and plates. Stab the inoculum down to the bottom of the deep in a clean. then just bring the loop straight up the slant. In the pure culture technique exercise. Pick only one colony or a bit of a colony. When streaking the agar. inoculate in a zig-zag pattern.Page 50 them. straight stroke. . There are 2 ways to inoculate the slant: If your goal is to identify the type of growth pattern. lift the lid just enough to insert the loop underneath: this will reduce contamination. Incubate the plate. in order to get your inoculating loop into it: DO NOT TAKE THE ENTIRE LID OFF. --ONTO AN AGAR SLANT: Place the loop with the bacteria into the slant tube. INTERPRETATION OF RESULTS: AFTER INCUBATION. --ONTO AN AGAR PLATE: 1. INOCULATING THE NEW MEDIUM --INTO A BROTH: The inoculum is just knocked around in the broth. 3. with your loop. Replace the lid and invert the plate. Lift the lid of the plate just a bit. 4. and against the sides of the tube in the broth. 2. This increases the surface area of the culture. While doing this. all the way down to the bottom of the slant. keep the loop horizontal and only streak the surface of the agar: DO NOT DIG INTO THE AGAR. starting at the bottom of the slant. If your goal is to have a luxuriant culture. if big. --INTO AN AGAR DEEP: Use the needle to inoculate the deeps or semi-solid agars.

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Why streak from the bottom of the agar slant medium up to the top in a straight line. Why use an agar plate to grow a bacterium rather than an agar medium slant or a broth medium? 4. How do you determine if an organism is growing in a broth medium? . What is the purpose of flaming the mouth of the tube? 5. rather than a back and forth wavy inoculation from side-to-side on the slant? 2. Of what use is it to know what kind of growth pattern on an agar slant or a broth medium an organism has? 3.Page 52 LABORATORY REPORT SHEET QUESTIONS: 1.

plant tissue. If it solidifies on you before you finish making your cultures. where a patient’s body fluid or tissue (skin. called the spread plate. Another technique. the medium will solidify very fast. In order to identify the bacteria and run antibiotic susceptibilities on the causative agent of the infection. Some of the TSA plates are already made: others will be made by you. urine) is sent to the microbiology lab for analysis. discard the plate in the autoclave bag. the 2 bacteria being used look different from each other when growing on agar plates. • Get another agar plate. coli and Serratia marcscens mixed together in TSB . REMEMBER…. the microbiologist must be working with a pure culture of the bacterium. If you see water running on the agar plate. it has to be disposed of (if not used yet. it can be placed back in the sterile media racks).. as is the pour plate technique. OBJECTIVES: Compare different agar plate isolation techniques. with the fluid inoculum being placed on top of the agar medium. Particularly in a medical setting. Agar’s solidification temperature is 42 degrees C. This requires that liquefied agar medium (sitting in a water bath) be kept above the solidification temperature. and particular uses. you can do 2 things: • Place the agar plate upside down in the 37C incubator with the top cracked. for example. pure cultures of the 2 bacteria. Two different types of agar plate isolation techniques will be used---streak and pour. That procedure will not be done today. The same mixed culture will be used for both methods. MATERIALS NEEDED: a culture of E. This also gives you a chance to see how agar plates are made. is used commonly for counts. blood. The inoculum is then spread around on the plate with a bent glass rod. or environmental samples) will be mixed. ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so.PURE CULTURE TECHNIQUES Page 53 Most specimens (from animal tissue. This exercise begins with a mixed culture of bacteria and will end with. A single gram of feces. the specimens will most often be mixed. has over 1010 bacteria and that gram would have over 20 different bacterial species. Differentiate among various colony morphologies. with a variety of bacteria (or other microorganisms). When sterilized in an autoclave or boiled. the medium will stay liquid until the temperature gets to 42 C. Each method has advantages and disadvantages. spinal fluid. It uses pre-made agar plates. hopefully. but its liquefaction temperature is 100 degrees C. Luckily. When that happens.

incubated at 25 C.. #2. carry it over to the water bath. incubated at 25 C Each table will make a set of pour plates from the mix. 4. and replace the top as soon as finished. the agar will be liquefied. Each table will make an extra streak plate from the mix. 5. aseptically transfer a loopful of the medium aseptically into tube #2. After adding the bacterial samples. Gently swirl the closed plate about 4 times in a large dinner plate-sized circle to spread the bacterial out well in the agar. Open the larger top of the dish RIGHT BEFORE pouring the agar into the dish. Be sure that the water just covers the agar. no higher.Page 54 water bath at 45 degrees C per person: 2 TSA plates (+ 2 more plates for 2nd session) per table: 3 TSA agar deeps 3 sterile Petri dishes 1 TSA agar plate THE PROCEDURES: Each student will make 2 streak plates from the mix. From tube #1. . DO NOT carry all 3 tubes at once: the others will solidy before you can inoculate them. Place the heater on high. The solidified agar deeps will need to be boiled and liquefied (agar medium will liquefy at 100 C). The water bath is set at 45 degrees C. 6. and #3 on the bottom of the dish. Pick up a loopful of your inoculum from the mixed bacterial culture and transfer it ASEPTICALLY into the first tube of liquefied TSA. Do this quickly. POUR PLATE TECHNIQUE (performed by the table) You will dilute the bacterial sample by transferring loopfuls of the specimen from media tube to media tube. Pour the entire contents of tube #1 into the Petri dish marked #1. Once at boiling temperature. 2. Remove 1 tube of liquefied TSA from the water bath and take it to your table. 1. DO NOT invert the tube of agar. Obtain sterile Petri dishes (make sure that cover stays on) and label as #1. Mix this tube #1 by rotating it quickly between your 2 hands: If done adequately. with fewer bacteria ending up in successive tubes. BE SURE THAT YOUR PETRI DISH IS SITTING RIGHT-SIDE UP. the agar is poured into sterile petri dishes and incubated. 7. there will be good mixing of the bacteria into the medium. It will take about 15 minutes for the hot agar medium to cool from 100 to 45 C. remove the hot agar tubes to a test tube rack. then cooled down BEFORE inoculating with the bacterial samples. Otherwise your bacteria will be cooked. Using the test tube holder. Place 3 TSA deep tubes into a glass beaker of water. incubated at 37 C. 3. and place into the tube racks in the water bath.

thereby diluting the sample as you move from section to section. lift the lid just enough to insert the loop underneath: this will reduce contamination. coli and Serratia marcescens on a TSA plate. keep the loop horizontal and only streak the surface of the agar: DO NOT DIG INTO THE AGAR. When streaking the agar. using 2 different streak techniques 1ST session: each student streak a plate from the mix. Mix tube #3. Pour the entire contents of tube #2 into the bottom of the Petri dish marked #2. Move the loop in a zig-zag pattern across the agar until 1/3 of the plate is covered. 11. . you might want to draw the 3 sections that you will streak inside of. finishing the first section. After incubation. Using a sterile agar medium plate. Remember to close the lid on the Petri dish between streakings. you will flame the loop between each plate section. Gently swirl the closed plate in large circles to spread the bacterial out well in the agar. on the back (bottom of plate containing agar medium) with a sharpie pen. Mix tube #2. 1. From this tube. Invert the agar plates when placing them at 25 C. 12. Pour the entire contents of tube #3 into the bottom of the Petri dish marked #3. check the 3 pour plates for colonies and their features in/on the agar. STREAK PLATE TECHNIQUE: (performed individually and as a table) Subculture a mix of E. Remember that you go into the mixed culture tube ONLY ONCE. Until you become well-acquainted with this procedure. 9. 2. and the table does an extra plate In this first technique. aseptically transfer a loopful of the medium aseptically into tube #3.Page 55 8. Mix the tube well by rotating it between your hands quickly. streak a vertical line straight down. While doing this. before the first section. Let the 3 plates solidify for at least 15 minutes before incubating. 10. 3. Gently swirl the closed plate in large circles o spread the bacterial out well in the agar. Pick up a loopful of your inoculum from the mixed bacterial culture.

5.Page 56 4. Rotate the plate about 90 degrees and spread the bacteria from the first streak into a second area using the same zig-zag spread technique. Each table will also make an extra streak plate from the mix. Lift the lid only enough to effectively streak. Invert the plate. Sterilize the loop again. . incubating it at 37 C. Sterilize the loop again. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria. 8. or 2) just holding the loop for a few seconds while it cools. Replace the lid. 7. You can do this by 1) sticking the hot loop in the agar at the edge of the agar away from the bacteria. Rotate the plate about 90 degrees and spread the bacteria from the second streak into the 3rd area in the same pattern. Second streak technique: This is identical to a procedure performed in an earlier lab. Each student will streak agar plates using these 2 techniques. This plate will be compared to one incubated at 25 C. Incubate the plate inverted at 25 C. Incubate the plate at 25 C. 6.

Compare your 2 streak techniques. shape. coli. Use your mixed culture from your agar plates to produce 2 pure cultures of the E. consisting of the 2 species that were merged together. Compare the streak plates incubated at 25 C and 37 C. coli and Serratia marcescens. 3. about the same size as E. 2. Pick a colony of each of the 2 different species of bacteria onto 2 new TSA agar plates. check the growth patterns of all plates. Subculturing in that situation will increase the chances of picking up another mixed culture. Serratia marcescens colonies will be pink/orange. Check the plates for purity the next period. ALWAYS pick a wellisolated colony when subculturing. check your streak technique or get your instructor to do so. Check pigmentation. Where are the colonies in the pour plates? Why? Why is there so much variation in colony sizes and shapes in the pour plates? Which method is best for accurate counting? Which method is the easiest to do? Which method gives easy access to the colonies---for subculturing to another medium? Which method has the greatest space between the colonies? 2. Stay away from merging colonies or close colonies.Page 57 2nd session: 1. . Incubate at 25 degrees C. b. Also. for feedback. INTERPRETATION OF RESULTS: 1. AFTER INCUBATION. coli colonies will be rather transparent and large. Compare the size. a. E. using your best aseptic streak technique. Check the agar plate culture of the mixed culture for 2 very different colony types. c. and location of colonies on the 3 kinds of plates.

or streak? Does it have good isolation? Why? . What difference was seen in the Serratia colonies grown at 25 C and 37 C? Why? 7. coli and Serratia marcescens mixed together. What is the purpose of flaming the mouth of the tube? 4. 5.Page 58 LABORATORY REPORT SHEET QUESTIONS: 1. When performing the streak technique. On the agar plate culture of E. and from the 3rd section back across the 2nd section? 2. Why use a streak plate to grow a bacterium rather than an agar medium slant or a broth medium? 3. Why are the colonies within the agar of a pour plate smaller than those on the surface of the agar? 6. spread. What procedure has been used to make this culture plate---pour. were the colonies in the first section of the plate the same size as those in the 3rd section? Explain why this would happen. why do you cross over back the 2nd streak section back into the 1st section.

Trying to pick a bit of one of those adjacent colonies increases the chances of picking up another mixed culture. dull (opposite of glistening). Eight obviously different colonies are numbered: some colony types recur in various areas of the plate (note # 3 and # 4). brittle/friable (dry. Two circles have been drawn around merging colonies. but distorted vision–like looking through frosted glass). where the species of the 2 colonies are different. breaks apart). In the identification of bacteria and fungi much weight is placed on how the organism grows in or on media. hard to get off). Features of the colonies may help to pinpoint the identity of the bacterium. translucent (almost clear. less than 1mm = punctiform (pin-point). opacity. therefore a colony constitutes a clone of bacteria all genetically alike. consisting of the 2 species that were merged together. an agar plate that has been exposed to the air and many different colony morphologies can be identified. Some pigments are water-soluble. opaque. edge. but also size. red. pattern. viscid (sticks to loop. A colony is defined as a visible mass of microorganisms all originating from a single mother cell. rough.BACTERIAL COLONY MORPHOLOGY Bacteria grow on solid media as colonies. others are not. and shine. ALWAYS pick a well-isolated colony when subculturing. Not only are pigment differences seen. rugose (wrinkled) CONSISTENCY: butyrous (buttery). WHOLE SHAPE OF COLONY SIZE OF COLONY (measure with a millimeter rule). glistening. iridescent (changing colors in reflected light) ELEVATION OF COLONY (turn the plate on end to determine height) SURFACE OF COLONY: smooth. Page 59 In the accompanying picture of a mixed culture. mucoid . If you take a large inoculum and place it in a tube of water or saline. do you see color? Do you see any pigment if the organism is growing in a broth medium? OPACITY OF COLONY: transparent (clear). it really can be important when identifying the bacterium. EDGE/MARGIN OF COLONY: magnified edge shape CHROMOGENESIS (pigmentation): white. etc. purple. buff. This exercise will help you identify the cultural characteristics of a bacterium on an agar plate---called colony morphology. Although one might not necessarily see the importance of colonial morphology at first.

coli and Serratia marscescens from last period THE PROCEDURES: 1. take a KimWipe and carefully remove the water from the cover. one light from above and one light from below the stage. .shtml OBJECTIVES: Describe features of colonies. 4. then quickly replacing the cover on the dish. The magnification is especially helpful for the study of elevation. surface. Streptococcus. 5. and a light behind the plate stage. 3. Place the plate RIGHTSIDE UP on the stage. There are 2 lights on these microscopes that you might find helpful. and Neisseria) agar plates of E. Chromobacterium. In order to determine CONSISTENCY. or even sometimes without them. or both. See variations in colonial morphology among various species of bacteria. 2. Bacillus. Make sure that the dish is right-side up. your culture will become contaminated. you need to use your inoculating loop or needle to pick up the colony and determine the consistency of the inoculum material as the loop leaves the agar medium. Use a dissecting/stereoscopic microscope for more detail.org/mentoring/project_ideas/MicroBio_Interpreting_Plates. If you see water condensation on the lid cover. Streptomyces.sciencebuddies. Use a plate which has well-isolated colonies. and edge. opacity.) There are 2 lenses on our scopes—10X and 20X: the black lens knob is on the right side of the head of the microscope. size. Interpreting Plates http://www. Micrococcus. leaving the petri dish cover ON (Otherwise. either using one at a time. Or you may want to use the Quebec colony counter since it has a magnifying glass. Look at the largest colonies with the naked eye to determine general shape and chromogenesis. MATERIALS NEEDED: agar plates of various bacteria (Pseudomonas. Two small black rotating knobs on either side of the base control the 2 lights.Page 60 Science Buddies.

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Page 62 LABORATORY REPORT SHEET QUESTIONS: 1. Which organism’s colony is easy to pick up using a inoculation loop/needle? Which one is difficult? 4. Why might the colonies of a particular bacterium vary when streaking onto different plate media? . Could you identify a contaminant bacterium on your pure culture plates? How? 2. Are the sizes of the colonies on a single petri-plate similar? 5. Define a clone. 3.

Therefore. (the reading on the pipet in picture is at 8. Place the pipette tip into the solution. you have inaccurately diluted the samples---either pipetting inaccurately or adding water to the tubes inaccurately. The following protocol is a step-by-step procedure to working dilution problems. for bacterial or fungal counts. or plaque-forming units. The purpose can be determination of bacterial. for viral counts). CFUs.1ml) Place the end of the pipette straight into the opening on the pi-pump (green pi-pump for 5 and 10ml. If done correctly. and includes some practice problems at the end. but accuracy is all important. rotate the pi-pump wheel so that the fluid ascends. OBJECTIVES: Become proficient with the use of the pipette. or viral counts (commonly called colony-forming units. Remember that the reading is taken at the bottom of the meniscus. It is a common practice to determine bacterial counts for both liquid and solid specimens--suspensions of E. it is important that each person understand how to use the pipette. fungal. © Peg Johnson In this little exercise. PFUs. Learn how to solve a dilution problem. no aseptic technique is required. and how to determine what dilution was produced. for a variety of purposes. coli in nutrient broth all the way to soil samples and hamburger meat. the last tube of each of the 4 dilution sets will be the EXACT shade of blue. how to read the pipette accurately. USE OF THE PIPETTE Be SURE that you know how to read the lines on the various sizes of pipettes.PIPETTING & DILUTIONS Page 63 Dilutions are used many times during the semester in the microbiology lab. If not. . blue pi-pump for 1ml). You will be starting out with a methylene blue-colored water and diluting it in various ways. Slowly move the wheel so that you correctly deliver the required amount. Identify different ways to dilute.

Dilution set 4: Transfer 0.Page 64 MATERIALS NEEDED: flask of blue water nonsterile pipettes . 4. Check the last 4 tubes of each set against each other to make sure that they are the same shade of blue. Prepare the following dilution blanks with tap water using a 10ml pipette and the green pi-pump---4. then 0. 9. slowly and accurately aliquot the correct amount into the new tube. 4ml. Make 4 sets of dilution tubes as seen in accompanying diagram. 9ml. 3ml. 2.9ml of water. Dilution set 3: Transfer 1ml of blue water into the 3ml of water. 5.5ml of water. • • • • Dilution set 1: Transfer 0. Dilution set 2: Transfer 1ml of blue water into the 4ml of water. then 0.5ml. b. and 10ml 8 nonsterile test tubes blue and green pi-pumps THE PROCEDURE: 1. 3. AND mix the tube contents (either roll the tube between your hands or pipet up and down a few times). Interpretation of your dilution tubes--a.5ml of blue water into the 4.5ml of tube 1 into the next tube of 12ml.5ml.1.1ml into the 9. 9.5ml.9ml. Use the most appropriate pipette size to transfer the solution.5ml of tube 1 into the next tube of 9. SOLVING DILUTION PROBLEMS: THE STANDARD FORMULA = _________colony count on an agar plate_________________ total dilution of tube (used to make plate for colony count) X amount plated . Fill a tube with methylene blue water. then 1ml of tube 1 into the next tube of 9ml water. 12ml. Record the dilution values of your tubes in the LAB PREPORT SHEET below.

and the amount of the dilution that was plated on the agar plate. average the counts together. Greater than 300 and less than 30 is a high degree of error. dilution factor for a tube = amount of sample amount of sample + amt. total dilution factor = previous dilution of tube X dilution of next container FOR THE ABOVE DILUTION SERIES: 0. STEP 1: Determine the appropriate plate for counting: Look at all plates and find the one with 30-300 colonies (or plaques). Determine the dilution factor for each tube in the dilution series.Page 65 To work the problem. There is nothing to calculate here: the value will be stated in the procedure.5 ml added to 4. Use the total dilution for the tube from where the plate count was obtained. each tube is a dilution of the previous dilution tube. preferably. you need 3 values---a colony count from the pour or spread plates. SO…. . or it will be given in the problem. of diluent in tube But after the first tube.5/5. If duplicate plates (with same amount plated) have been made from one dilution..5ml = 0.0 = 5/50 = 1/10 for 1st tube 1ml added to 9ml = 1/10 for 2nd tube previous dilution of 1/10 (1st tube) X 1/10 (2nd tube) = total dilution of 1/100 STEP 3: Determine the amount plated (the amount of dilution used to make the particular pour plate or spread plate). STEP 2: Determine the total dilution for the dilution tubes: Dilution = amount of specimen transferred divided by the [amount of specimen transferred + amount already in tube]. a dilution factor for the dilution tube from which the countable agar plate comes. Air contaminants can contribute significantly to a really low count and a high count can be confounded by error in counting too many small colonies. Multiply the individual dilution of the tube X previous total dilution To calculate this dilution series: Determine the dilution of each tube in the set.

000/ml . 45 colonies = 1/103 X 1/10 45 X 104 = 4. 3.Page 66 SOLVING ABOVE PROBLEM: 1.cannot multiply fractions and decimals together).1ml (convert to 1/10 . 2. The countable plate is the one with 45 colonies.5 X 105 (scientific notation) OR 450. The amount used to make that pour plate = 0. The total dilution of the 2nd tube from which that pour plate was made = 1/102 X 1/10 (equals 10/100) = 1/103.

For the dilution tubes with colored water. fill in the following values SET 1 tube 1 tube 2 SET 2 tube 1 tube 2 SET 3 tube 1 tube 2 SET 4 tube 1 TOTAL DILUTION 1/10 1/100 TOTAL DILUTION _______ _______ TOTAL DILUTION _______ _______ DILUTION ________ 5. . What is the meniscus? 2.Page 67 LABORATORY REPORT SHEET QUESTIONS: 1. If you transfer 0.1ml of a sample into a 99. what is the dilution? 3. How much fluid is IN the pipette at right? 4.9ml saline blank.

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the amount of transmitted light decreases as the cell population increases. Escherichia coli. Thus. Although the two methods are somewhat similar in the results they yield. For example. namely. coli used in this exercise? When working with large numbers and a short time frame. 10-4 to 10-10) is normally plated because the exact number of bacteria is usually unknown. there are distinct differences. The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass). The two most widely used methods for determining bacterial numbers are the standard.. That is. coli cells in a bacterial culture can be easily determined by spectrophotometry). plate count method and spectrophotometric (turbidimetric) analysis.g. A wide series of dilutions (e. coli has a generation time at 37C of 20 minutes. the number (biomass) of viable E.e. Become proficient at performing a standard plate count and determining bacterial counts in a sample. OBJECTIVES: Correlate absorbance value for a bacterial suspension with an accurate bacterial count. Greater accuracy is achieved by plating duplicates or triplicates of each dilution. E. and more than 300 colonies on a plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs). or viable. MATERIALS NEEDED: per table (exercise performed by table) 24-hour 10ml nutrient broth culture of Escherichia coli . although we will not be doing that in this exercise. Why Is E.COUNTING BACTERIA Page 69 Many studies require the quantitative determination of bacterial populations. The transmitted light is converted to electrical energy. and this is indicated on a galvanometer. The procedure for the spectrophotometer use is at the end of this exercise. one of the most reliable microorganisms is one that has been used in previous experiments. Increased turbidity in a culture is another index of bacterial growth and cell numbers (biomass). it reproduces very rapidly and is easy to quantify (i. Thus. Become proficient at dilutions. Fewer than 30 colonies are not acceptable for statistical reasons (too few may not be representative of the sample). the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony (CFU). called absorbance or optical density. The reading. This method is faster than the standard plate count but is limited because sensitivity is restricted to bacterial suspensions of 10 7 cells or greater. indirectly reflects the number of bacteria.. By using a spectrophotometer. the number of colonies should give the number of bacteria that can grow under the incubation conditions employed. dead and alive. the final plates in the series should have between 30 and 300 colonies.

Remove one agar pour tube from the 48 to 50C water bath. 7. When the deeps are liquefied. and 10-8.COLI FOR THE NEXT SECTION! Page 70 START BOILING WATER TO LIQUEFY NUTRIENT AGAR DEEPS before starting this section. 2. This third dilution represents a 10-6 dilution of the original sample. the initial dilution is made by transferring 1 ml of E. Immediately after the 10-2 dilution has been shaken. This serves to distribute the bacteria and break up any clumps. 10 -6. Shake the 10-4 dilution vigorously and transfer 1ml to the third 99ml blank. this second blank represents a 10-4 dilution of the original sample. Repeat the process once more to produce a 10-8 dilution. 5.1 ml to another petri plate. Since this is a 10-2 dilution. Label four tubes of saline 10-2. Carefully remove the cover from the 10-4 petri plate and aseptically pour the agar into it. coli sample to a 99ml sterile saline blank (figure below. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands and rotating quickly to create a vortex. 4. Shake the 10-4 dilution again and aseptically transfer 1.4 sterile 99-ml saline blanks 1-ml pipettes with pi-pump 6 petri plates 6 agar pour tubes of nutrient agar (plate count agar) 48 to 50C water bath boiling water bath Bunsen burner 6 micro-cuvettes and rack 1 micro-cuvette holder spectrophotometer 4 tubes of 5ml nutrient broths 4-5ml pipets and pi-pump THE PROCEDURES: STANDARD PLATE COUNT BE SURE TO SAVE YOUR ORIGINAL TUBE OF E. Using aseptic technique. The agar and sample are immediately mixed gently moving the plate in a figure-eight motion or a . 1. 6.0 ml to one petri plate and 0. Label the bottom of six petri plates 1-6. This is a 1/100 or 10-2 dilution. they can be placed into the water bath to cool so that the pours can be made without killing the bacteria. 3. Cooling takes about 20 minutes. 10-4. Do the same for the 10-6 and the 10-8 dilutions. uncap it and aseptically transfer 1ml to a second 99ml saline blank.

After the pour plates have cooled and the agar has hardened. coli to the first tube of NB. a. and so on until the last of the 4 tubes has 5ml added to it. but you can guesstimate the amount by eyesight. and 1/16 dilutions. c. Save BLANK to re-standardize the machine to infinity absorbance and zero absorbance before each reading because the settings tend to drift. coli and four tubes of the sterile NB in a test-tube rack. Place in cuvette holder and read. close the cover and read. they are inverted and incubated at 25C for 48 hours or 37C for 24 hours. Pipette 1ml of the original bacterial specimen into a second micro-cuvette. 8. A Quebec colony counter should be used. The wavelength is preset somewhere between 550600nm. Transfer 5ml from that tube to the next tube. 2. 1/4. e. The cuvettes will look like either of the 2 shown to the right. 1/8. Place into the black cuvette holder (red line towards you). When read. 10. Transfer 5ml of E. Pipette 1ml of the sterile NB into one of the micro-cuvettes. Repeat this process for the remaining five plates. The microcuvette must contain1ml for the spectrophotometer to read the fluid. The lined or etched sides of the cuvettes face you. Notice the arrow on picture above showing where level of fluid must be. Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC). Plates with fewer than 30 colonies are designated too few to count (TFTC). TURBIDIMETRY DETERMINATION OF BACTERIAL NUMBERS THIS SECTION DOES NOT HAVE TO BE DONE ASEPTICALLY! 1. Use four of these tubes (tubes 2 to 5) of broth to make four serial dilutions of the culture (figure 2). d. The directions for spectrophotometer use are BELOW. Put the ORIGINAL tube of E.Page 71 circular motion while it rests on the tabletop. discard micro-cuvette into bleach . At the end of the incubation period. Each tube of NB contains 5 ml of sterile broth. 3. Obtain the 6 micro-cuvettes. with the clear sides facing the light source. Standardize the spectrophotometer as directed. thoroughly mixing the tube afterwards. select all of the petri plates containing between 30 and 300 colonies. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to liquefied agar. number of colonies (CFUs) = # of bacteria/ml dilution X amount plated 11. Count the colonies on each plate. The BLANK used to standardize the machine is sterile nutrient broth: it is called the BLANK because it has a sample concentration equal to zero. 9. DO NOT change it! (Instructor will set the wavelength) b. Record your results. These tubes will be ½.

2. along with the dilutions that they came from. 5. 1/4. 1/8. DATA COLLECTION dilutions absorbance (X) # of bacteria (Y) original E. 3. 4. coli 1/2 1/4 1/8 1/16 1. Calculate the number of bacteria in the original tube of E. calculate the colony-forming units per milliliter for each dilution. and place that value in the top right cell of the table. o Mix the dilutions before pipetting into the micro-cuvette to read absorbance. o Re-standardize between readings to account for drift. Next pipette the ½ dilution into the third cuvette and read it. coli. YOU MUST HAVE EQUAL INTERVALS ALONG BOTH X AXIS AND Y AXIS. Repeat this with the ¼. 4.Page 72 container on your table. Record your values. Calculate the approximate numbers of bacteria in the ½. Things to remember : o Place micro-cuvette with parallel lines facing you into cuvette holder. Plot these 5 coordinates on a graph. o Read to the nearest thousandth (0.001) on the absorbance digital display. Using the plate count data. 1/8. Fill in your absorbance values for the 5 tubes read in the spectrophotometer. . Here is an example of a graph. using EXCEL software (it is available in the computer labs). and 1/16 by halving the number in the cell above. and 1/16 dilutions. The DIRECTIONS on how to use the software is at end of exercise. o Close the hatch when reading the spectrophotometer so no light enters.

coli 1/2 1/4 1/8 1/16 absorbance (X) # of bacteria (Y) 2. can it also be used for another bacterium like Staph? 4. 6. Give the formula for determining bacterial counts. Give the bacterial count per milliliter of E. How is transmission different from absorbance? 5.Page 73 LABORATORY REPORT SHEET QUESTIONS: 1. If you have a graph for E. coli suspension in the original culture tube. . DATA COLLECTION: dilutions original E. coli. Why do a standard plate count when running turbidity values the first time? 3.

With the cuvette chamber empty and the cover closed. A clear solution will allow almost all of the light through. then through the sample and onto a light-sensitive phototube. a light shines through a filter (which can be adjusted by controlling the wavelength of light). . A clear tube of water or other clear solution is the BLANK and has zero absorbance. which measures what fraction of the light passes through a given solution and indicates on the absorbance display the amount of light absorbed compared to that absorbed by a clear solution. Pressing the MODE button will toggle to the TRANSMITTANCE mode. This produces an electrical current. use the power switch knob on the LEFT to adjust the digital display to 0% Transmittance. Set the display mode to ABSORBANCE by pressing the MODE control key until the appropriate red LED is lit. 1. The absorbance meter measures how much light has been blocked by the sample and thereby prevented from striking the phototube. A graph of absorbance vs. The amount of absorbance can be determined by using a spectrophotometer. 2. (Instructor will tell you what the wavelength should be so you can double check the displayed setting. . The following numbered steps coincide with the numbers on the spectrophotometer at right. The amount of substance in the solution is directly proportional to the absorbance reading. concentration will produce a straight line.Page 74 USE OF THE SPECTROPHOTOMETER Light entering a cloudy solution will be absorbed.) The 1st white button (labeled #3) on the Display panel will toggle between MODE and TRANSMITTANCE. 3. Inside. DO NOT change it. The wavelength and filter have already been set.

With the right-hand knob. Then place into the cuvette chamber. It is necessary to reset the machine to infinity absorbance and zero absorbance before each reading because the settings drift a bit. BOTH infinity and zero absorbances must be re-set. 6. adjust the display to read zero absorbance.Page 75 4. 5. 8. the lined sides will FACE you). Remove the BLANK from the cuvette chamber. Wipe it free of moisture and fingerprints with a KIMWIPE and insert it into the cuvette holder with the "V" mark facing you (OR we may have the cuvettes which have parallel lines on the sides---if so. Close the cover. Fill a micro-cuvette with 1ml of NB to serve as the BLANK. . 7. and insert the holder and microcuvette with your sample into the chamber. Read the absorbance value directly from the digital display.

Using the diagram on this next page as a reference. 3. Click on cell A2 with the left mouse button (LMB) and drag the mouse to highlight the area containing all the data points (go down to cell B8). enter the data of bacterial numbers and absorbance readings of your table. Each box in the grid is called a cell. Numbers data 1. Sara Perez-Ramos. (Leave the chart sub-type alone: the top chart should be highlighted. Each cell has an “address” made up of a letter indicating the column the cell is in and a number indicating the row the cell is in. Double click on the Microsoft Excel icon to open the program. then on “Chart”. Double click on cell A1 to start entering information. the upper left cell is A1. You can also choose to make some cosmetic touches (changing the size of fonts.Page 76 Plotting Data Using Microsoft Excel Prepared by Dr. Now you will learn how to set-up the axes. . Select “XY(Scatter)” and click on “Next”. Once the data is highlighted. A box with graph options will appear. modified by Jackie Reynolds Plotting Best-Fit Lines of Absorbance vs.g. The screen should look like this: 4. You will also command the computer to draw best-fit lines and calculate the slope of each plot. look on the upper toolbar and click on “Insert”. 2.) Your data will appear already plotted on an x-y axis system. write the title of the graph and write the legend.…) on your graph before printing. e. You will see a grid. colors of the plot etc.

7. Find “Series” towards the top of the graph and click on it. Click on “Next” click on the button titled Place chart as object in sheet 1 (NOT as new sheet). Before clicking OK. Press the RIGHT mouse button. As you do this. Click on “OK”. e. The graph will show a best-fit line. Make sure that in the window “Chart Title” your graph is titled (e. for example). Click on “Display equation on chart”. Click on “Next”.5. Another box will appear. Select and click on “Add Trendline”. Using Turbidemetry to Guesstimate Bacterial Numbers”). Select Linear (should already be highlighted). Click on space below Value(X)Axis and type: Absorbance c.g. Page 77 6. You can alter its size by clicking the LMB on the black squares around the graph and dragging the mouse with the LMB pressed down. Click on “Major Gridlines” on the x-axis and y-axis. Now you are going to add the line that goes through the data points (trendline).) f. Click on “Axes” (upper left of graph) and make sure that Value(X)Axis and Value(Y) Axis have a √ . 8. On the graph you should see an equation which gives you the value of the slope for the line). a. Click on space below Value(Y)Axis and type: Bacterial Numbers (X 106) d. Click on the graph to make sure that the black squares around the graph are there. Another box will appear with options to modify titles on the graph. . Move the pointer to any of the data points of a particular series (any ∆. information about this data point will appear on the screen. To the right of this there is an empty space for you to write the NAME of this plot: click on this empty box and type “Absorbance vs. go to “Options” on the top of the box. b. Click on “Finish”. Bacterial Numbers”. A box with choices appears. Click on “Gridlines” and add lines. Your graph will appear on the screen as shown below. Click on “Legend” and choose the location of the legend (I chose bottom). “Series1” should appear highlighted.

Page 78 THE GRAPH SHOULD LOOK LIKE THE PICTURE ABOVE. Click on “Format Axis”. Select and click on “Format Plot Area”. 11. . until you are satisfied with the product. Click on “Font” and reduce the font size (I chose 8). Select and click on “Format Data Labels”. If it’s not ready try more cosmetic changes as desired. Click the RIGHT mouse button on top of the scale numbers. Changing the size of the axis label: Click on the desired label to change (e. Go to “File”. Name the file: EXAMPLE: turbidimetry. c. Do this for the scale on the other axis also. Insert your 3½” diskette in the computer. Click on “File”. Click on The legend box until you see black squares around its box. click on “Print” and “OK”. To change the color of the graph background: Press the RIGHT mouse button while the cursor is on the gray area. Click on “Font” and reduce the font size (I chose 10). Click on “OK”. Click the RIGHT mouse button. You can also alter the size of the font for the legend items. the x axis label that says “Time in seconds”). 9. Do the same for the other axis. Click the RIGHT mouse button. Click on “OK”. Select and click on “Format Axis Title”. “Save As” and choose A: 3½” drive as your destination. and change the font size as desired (I chose 10). Changing the position of the equation: Click on the equation and drag that box to the desired position. Press the RIGHT mouse button.xls. To edit the Legend box: Click on the Legend box. select and click on “Clear”. If it is ready. Go to the top toolbar. b. You may also want to alter the size of the numbers on the scale. 10. You may want to choose a white background if your printer is black and white. then set the font to the desired size (I chose 8). e. Changing the size of the font used for the equation: Click on the equation to be changed.g. BUT WITH A LINE AND THE SLOPE FORMULA. Click on top of 1/2x [enzyme](for example) with the RIGHT mouse button until black squares around it appear. you want to be sure that all your work is saved. “Print Preview” and check if the graph is ready to print. Cosmetic changes for graphs: a. Do this for all the items in the legend. d. Select “Format Legend”. Before proceeding any further.

and the type of microorganism. represented by the formula: [H+] = moles of H+/liter Page 79 The value represents a base 10 logarithm. causing electrons to be pulled off of DNA molecules and oxidizing it. However. Hypertonic environmental conditions cause the cells to dehydrate as water osmoses out of the cell. hence a pH of 7 is neutral. etc. Less than 7 is acidic (the more extreme the number. That is. Like your human cells. can tolerate hypertonic conditions produced by high salt or high sugar concentrations.). Radiation is often used to control microbial growth (rooms. Two major forms of electromagnetic radiation can be mutagenic—ionizing radiation (X-rays.ENVIRONMENTAL CONDITIONS & BACTERIA GROWTH Many environmental conditions can affect microbial growth---temperature. sugar. most bacteria optimally grow in isotonic conditions. Acidophiles and alkalinophiles are the extreme microorganism groups. and barometric pressure. The flow of water is controlled by the osmotic pressure of the environment. gamma) and ultraviolet. particularly fungi. Obviously you will have to remove the tops of the plastic petri dishes when exposing the bacteria to the UV. and pH therefore defines a logarithmic scale of acidity. Water naturally dissociates into H+ and OH− ions with equal concentrations of 1×10−7 M. although they do not really require the salt to grow. the less the H ion). The difference in numbers on the pH scale (0 to 14) is 10-fold. its concentration. Cells have repair mechanisms that . In the following exercise. Those organisms that require high salt are called halophilic.g. the greater the H ion).). These thymine dimers affect the replication of the DNA in the dividing cell. e. etc. many microbes. the degree of inhibition of growth will depend on the type of dissolved particles (salt. citrus fruit decay. the most germicidal being UV-C (because it penetrates the best). Ionizing radiation has much greater kill ability because of its penetration (short wavelength). The killing ability of radiation is due to mutations in the DNA produced by the radiation. Fungi often can live at lower pH conditions. There are different kinds of ultraviolet. and that is determined by the concentration of solute molecules. some requiring concentrations of 15-20% NaCl. or plastic. foods. PH is the value determined by the concentration of hydrogen ions in a liquid. pH 8 is 10X more basic than pH 7 and pH 3 is 100X more acidic than pH 5. Most bacteria live within the pH range of 4-9. Generally. Ionizing is the more potent since it is much shorter wavelength. Many bacteria can tolerate high salt concentrations (halotolerant). osmotic pressure. packaged products. pH. Ultraviolet radiation exerts it effect by causing adjacent pyrimidine nitrogenous bases (commonly thymine bases) to bond with each other. while higher than 7 is basic (the more extreme. radiation. glass. meaning that the strand cannot effectively attach to its complementary strand. you will look at the effect of various salt concentrations on bacterial growth. Osmophilic microorganisms tend to be fungi. Ultraviolet radiation does not generally penetrate clothes.

Staph aureus. the cell will not live. Incubate the plates at room temperature. producing an enzyme that uses energy of visible light to split the thymine dimmers The latter method is called photoreactivation. 3. trying to use the same amount of inoculum on each plate and inoculated the same way for consistency.Page 80 counteract these mutations. and 10 for each organism UV radiation exercise 4 X 6 index cards 5 TSA plates 8 sterile cotton swabs brown paper bag UV lamp (bulb should be positioned 18 inches above the table top) TSB cultures (same density of cells in each) of Pseudomonas aeruginosa. coli. MATERIALS NEEDED: per table OSMOTIC PRESSURE exercise 1 TSA plate each of 0. and 10% NaCl concentrations PH exercise 1 TSB pH 3. Each table will obtain one TSA plate of each salt concentration and divide the plate into 3 sections. S no growth +1 very light growth +2 medium growth +3 heavy growth . and a spore suspension of Bacillus subtilus (for UV study only) THE PROCEDURES: OSMOTIC PRESSURE (each table uses all 3 organisms) different inoculation patterns 1. excising the incorrect section of the DNA with enzymes 2. regular Bacillus. Streptococcus lactis. 7. Bacillus subtilus. and determine how the damage can be reversed. Serratia marcescens. Inoculate a section of each agar plate with a loopful of your organism (E. OBJECTIVES: Identify the effects of ultraviolet radiation on bacterial growth. but if the mutagenic agent is producing the mutations faster than can be fixed. Quantify the amount of growth by comparing the density of the organisms on the plates. Identify the effects of osmotic pressure on bacterial growth. coli. 4.5% (regular TSA). INTERPRETATION: During the next lab period you will determine the effect of salt on the growth of your bacterium. The cell repairs the mutation in different ways: 1. Identify the effects of pH on bacterial growth. and Staph on the sections of the plate). Alkaligenes fecalis. 5%. Micrococcus luteus. E. 2.

7. S no growth +1 very light turbidity +2 medium turbidity +3 heavy turbidity PH . Each table will obtain 3TSB tubes of pH 3. Streptococcus lactis. 7. 3. on top of the petri dish with cover on it. You should be able to get all of the plates directly under the lamp. 3. QUICKLY place each plate into a paper bag. INTERPRETATION: During the next lab period you will determine the effect of pH on the growth of your bacterium. 1 covered with lid) 5 minutes 10 minutes (Place the index card over ½ of the agar plate at a right angle to the swab lines. exposure time. SHAKE each broth and hold all tubes in one hand up to the light. Inoculate each TSB with a loopful of your organism. Place those 2 plates (with lids now on) under a regular lamp for 10-15 minutes. and 10. remove the card. 6. Remove the each plate from the UV. 8. except the 10 minute exposure (see directions below). Take your plates to the UV lamp and place on the table top (18 inches below the bulb). Marking the time on your watch or a clock as time ZERO). a total of 6 plates. Remove the plate cover when ready to start timing) WARNING: Do not look directly into the UV light as it can cause damage to your eyes. exposure times for plates: 10 seconds 60 seconds (2 plates—1 exposed. (each table will use 1 organism) 1. Your table will use Alcaligenes faecalis. Bacillus subtilus or Escherichia coli. 2. and whether it will be used for photoreaction or has a lid. Use the swab to smear the culture all over the ENTIRE plate of agar. and cover the plate with the Petri dish lid. Do the same with all of the other agar plates. 2. Dip the swab in your assigned culture. Do NOT remove the tops of the petri dishes until READY to expose to UV. Incubate all plates at room temperature 25 degrees C for 24 hours. 5. remove the petri dish cover and expose the plate to the UV for the assigned amount of time. Two plates will be left out for photoreactivation—a 60 second and a 5 minute.Page 81 ULTRAVIOLET RADIATION (each table will use1 organism) 1. 4. Do not expose your direct skin to the UV. Quantify the growth by comparing the densities. Streak back and forth in a DENSE zig-zag pattern repeatedly to cover every bit of agar. Label your 8 plates with the name of the bacterium.

or +3 to quantify. 10 min. +2. Record the results from your pH exercise. using -. Record your data from the UV exercise. +1. species pH 3 pH 7 pH 10 Alcaligenes faecalis E. species 0. +2. Record your results from the osmotic pressure exercise. 15 min. coli Strep lactis Bacillus subtilus 3. using -. Include data on the other organism from the class results. UV EXPOSURE TIME species 10 sec 60 sec 5 min. What do you call an organism that prefers acidic environments? 4. Which of the organisms grew best in alkaline pH? Which bacterium was the most halophilic? 5. coli Staph 2.5% NaCl 5% NaCl 10% NaCl Bacillus E. Include data on the other organisms from the class results. .Page 82 LABORATORY REPORT SHEET QUESTIONS: 1. or +3 to quantify. +1.

EFFECT OF TEMPERATURE ON BACTERIA GROWTH

Page 83

Bacteria and fungi can grow across a large spectrum of environmental conditions. Even though the bacterium may grow well in the human body at 37 C at pH 7 conditions, it may also be able to withstand out-of-the-ordinary pH, temperature, and osmotic pressure. Basically, there are 3 large groups of microorganisms with respect to their temperature preference---mesophiles, thermophiles, and psychrophiles. Animal pathogens are mesophilic, growing well in the range of 20-45C. Psychrophiles have an optimal range below mesophiles (even below freezing), while thermophiles grow at temperatures over 45C. Interestingly, many organisms grow differently in different temperatures, and you have seen evidence of this already in lab. In an earlier exercise, you should have noticed that the orange-pigmented Serratia marcescens pigments at 25C but not at 37C. The prodigiosin pigment, a waste product produced during certain metabolic pathways, is temperaturedependent. Also, some fungi will change structure at these 2 temperatures. For example, Candida albicans, the cause of thrush and yeast infections, will produce unicellular yeast cells at 37C, but at 25C will produce chlamydiospores and hyphal filaments. Although endospore-forming bacteria such as Bacillus can endure very high temperatures, these bacteria do not optimally grow in those temperatures, hence the term thermoduric. The spore wall composition and its dehydrated state make it very resistant to harsh environmental conditions. OBJECTIVES: Recognize the effects of temperature on bacterial growth and spore resistance. Identify the temperature group based on bacterial growth pattern.

MATERIALS NEEDED: per table Optimal growth - 5 TSB tubes (3ml) per organism Resistance to Extreme Temperature - 8 TSB for E. coli and Bacillus spore suspension 1ml pipets and pi-pumps 2 TSA plates TSB cultures (same density of cells in each) of Pseudomonas, E. coli, Micrococcus luteus, Bacillus stearothermophilus, Bacillus subtilus spore suspension of Bacillus subtilus (for extreme temperature section ONLY) PROCEDURE 1. OPTIMAL GROWTH TEMPERATURES – each table uses 1 bacterium at all temperatures a. Inoculate 5-3ml TSB tubes with a loopful of the test organisms--- Bacillus stearothermophilus, Pseudomonas, E. coli, or Micrococcus. You will use 5 tubes per organism. Be sure to label each tube with temperature and the name of the organism.

Page 84 b. Incubate each tube at a different temperature: 10oC (fridge) 25oC (room temperature shelves) 30oC (small yellow incubator) 37oC 45oC (small metal incubator) c. INTERPRETATION: Determine the optimal temperature at which your bacterium grows. To do this, you need to quantify the amount of growth by comparing the turbidity (AFTER shaking the contents) in all 5 tubes. S no growth +1 very light turbidity +2 medium turbidity +3 heavy turbidity

2. RESISTANCE OF CELLS AND SPORES TO EXTREME TEMPERATURES a. Inoculate 4 tubes of TSB broth each with a 0.1ml inoculum from the E. coli culture and another 4 tubes with the Bacillus spore suspension. Label the tubes as control, 50, 70, and 100, plus the type of culture on each tube. b. Place each tube in the water bath equilibrated to one of the temperatures, or in a boiling water bath. Incubate for exactly 10 minutes. Be sure that the water is deep enough so that the broth of the tube will be totally immersed. c. After 10 minutes exposure to the temperature, streak out a loopful of each of the cultures from each temperature on a separate TS plate and incubate at room temperature.

control 70C

50C 100C

control 70C

50C 100C

Use a zig-zag or a straight line inoculation, but be CONSISTENT between sections.

E. coli

spores of Bacillus

d. INTERPRETATION: Determine the effects of various temperatures on a vegetative cell suspension and the spore suspension. To do this, you need to quantify the amount of growth by comparing the amount of growth on the agar plate. S no growth +1 very light growth +2 medium growth +3 heavy growth

Page 85

LABORATORY REPORT SHEET
QUESTIONS: 1. Fill out the table below by PREDICTING where each type of organism would optimally grow. Use a + sign to denote growth. Organism 10 °C Psychrophile Mesophile Thermophile Hyperthermophile 25 °C 30 °C 37 °C 45 °C

2. Record your results by adding 0, +1, +2, or +3 for growth. Species E. coli Micrococcus Pseudomonas Bacillus stearothermophilus 10 °C 25 °C 30 °C 37 °C 45 °C Classification based on Optimal temperature

3. Record the results of the extreme temperature exercise---- 0, +1, +2, or +3 for growth. Temperature Control 50 70 100C E. coli cells Bacillus spores

4. What is the difference between a thermophile and a hyperthermophile?

5. How does extremely high temperature kill vegetative cells?

Page 86 .

You will need to bring your own antiseptic or disinfectant chemicals for this lab. Label the quadrants as follows: control. In the second exercise. MATERIALS NEEDED: per table 4 sterile glass rods ("thermometers") 1 TSA plate 4 test tubes (3 for water. Identify which categories of chemicals are most effective. Compare kill ability of a chemical at different exposure times. A glass rod will simulate a thermometer. disinfectants are harsher on human tissue. the chemicals that you are familiar with are not sterilizing agents.EVALUATION OF ANTIMICROBIAL CHEMICALS Page 87 Antiseptics and disinfectants are chemicals which kill or inhibit growth.). although they are not always more effective than antiseptics. 15 seconds. etc. but not 100% kill. Divide the TSA plate into 4 quadrants with a marker. This lab will give you the chance to evaluate your household chemicals. floors. Antiseptics are used on tissues. Generally. b. Fill 3 tubes with 5 ml of deionized water . coli 2 TSA plates sterile paper disks 4 tiny beakers antiseptic and disinfectant chemicals (4-5 per table) forceps and beakers of ethanol THE PROCEDURE: Antiseptic Action on Oral Bacteria Each table will test one particular antiseptic against oral normal flora.” Be sure to include the name of the chemical your table is using as well as the specimen type. OBJECTIVES: Compare the activity of antiseptic and disinfectant chemicals. 1. Be sure to write down the active ingredient in the chemical: it will be listed on the label. Four different “thermometers” will be exposed to a disinfectant or antiseptic for a certain amount of time. we will look at the effectiveness of a disinfectant or antiseptic against mixed bacterial flora in your mouth. and “1 minute. and 1 tube for the antiseptic) A small beaker with tap water large bowl of bleach (to discard “thermometers”) TSB cultures of Staphylococcus epidermidis and E. However. whereas disinfectants are for inanimate objects (utensils. tables. Sterilizing agents have a 100% kill. 30 seconds. ALL glass rods from mouth or from bacterial culture go into a bleach container at your table. Prepare materials for the exercise: a.

let excess water drip off the tip. Remove the other glass rods and expose each to the same chemical for the designated time period. BELOW is a schematic of this activity. place the 1st rod in your first tube of sterile water and stir it around for a couple of seconds. THIS IS THE CONTROL—no exposure to a chemical. After 3 minutes. • Place rod #4 into your tube of chemical for 1 minute. 5. 4. 3. • Place rod #3 into your tube of chemical for 30 seconds. and streak the tip of the rod on the control sector of the TSA plate. Fill 1 tube with your chosen antiseptic. Place the 4 glass rods into your mouth for 3 minutes. Remove the rod from the water. • Place rod #2 into your tube of chemical for 15 seconds. As you did with rod #1. Incubate the TSA plate at 37C until the next lab period. and then streak a quadrant of the TSA plate with the rod tip. . 6. stir rods 2-4 in its own tube of sterile water. Be careful that the inoculum does not enter the other sector of the plate. let the excess water drip off (you do NOT want the fluid to run from one quadrant to another quadrant). 2.Page 88 c.

LABORATORY REPORT SHEET QUESTIONS: 1. Repeat the procedure with the other chemicals. pouring a small amount of each liquid into a tiny beaker.Page 89 THE PROCEDURE: Comparison of Antimicrobial Chemicals Inoculate the plates of TSA with the bacterial species by swabbing the cultures well over the entire surface of the plate. Incubate the plates at room temperature. If there is no zone around the disk. much growth = +2 other chemicals used? time control 15 seconds 30 seconds 1 minute control 15 seconds 30 seconds 1 minute control 15 seconds 30 seconds 1 minute 70% EtOH 3% H2O2 mouthwash A mouthwash B . The paper disc is about 6mm diameter. Record the results for the entire class in the table on this handout. little growth = +1. Label the bottom of the agar plates with the names of the chemicals being evaluated. Using alcohol-flamed forceps. Pick 4 chemicals to test. Be sure that you use the millimeter measurement on the metric ruler. Use the following quantitative criteria to determine effectiveness of the chemical at different exposure times----no growth = — . pick up a sterile paper disk and dip it halfway into the chemical. no growth = — . The 2 organisms will be tested with the same chemicals. INTERPRETATION: Antiseptic Action on Oral Bacteria Look at each quadrant of your TSA plate and determine whether there is growth or no growth. Each table should have 2 agar plates with the 2 species of bacteria. much growth = +2 Comparison of Antimicrobial Chemicals Measure each zone of inhibition around the paper discs. Record data from your own chemical and specimens as well as other tables’ data. little growth = +1. then place the disk on the inoculated media. 25oC. or if the zone size is very small---less than 10mm—record as 0.

4.Page 90 2. coli 3. How long an exposure time does one need for kill of the bacteria? . To which chemical is this bacterium most resistant? 5. Name of chemical Active ingredient ZONE DIAMETER (millimeters) Staph E. List the 3 chemicals used in lab that had the greatest killing ability. Record the results for the various chemicals used in this exercise.

Staphylococcus aureus is a common normal flora bacterium found in the body. But the K-B is still used in some labs. or intermediate. called the disc diffusion test. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug. that is. The Kirby-Bauer test for antibiotic susceptibility. or used with certain bacteria that automation does not work well with. MATERIALS NEEDED: per table 4 Mueller-Hinton agar plates 24 hr old cultures of Staph. It is also likely that if antibiotic sensitivity tests were run on these isolates. If one isolated this bacterium from 5 different people. there will be NO growth in the immediate area around the disc: This is called the zone of inhibition.KIRBY-BAUER TEST FOR ANTIBIOTIC SUSCEPTIBILITY Page 91 A true antibiotic is an antimicrobial chemical produced by microorganisms against other microorganisms. The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs are placed on top. is a standard that has been used for years. It has been superseded in clinical labs by automated tests. the 5 isolates would likely be different strains. Bacteria respond in different ways to antibiotics and chemosynthetic drugs. Mankind has made very good use of these antimicrobials in its fight against infectious disease. The Mueller-Hinton medium being used for the Kirby-Bauer test is very high in protein. The zone sizes are looked up on a standardized chart to give a result of sensitive. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. E. even within the same species. Bacillus subtilus. the results would vary against the different antibiotics used. coli. For example. slight genetically different. Many drugs are now completely synthetic or the natural drug is manipulated to change its structure somewhat. Strep fecalis sterile swabs antibiotics ethanol + forceps Pseudomonas aeruginosa Kirby-Bauer plate for demo guidelines chart for interpretation of antibiotic susceptibility . The MIC is currently the standard test run for antibiotic sensitivity testing because it produces more pertinent information on minimal dosages. resistant. the latter called semisynthetics. OBJECTIVES: Determine the susceptibility of various bacterial species to various antibiotics and synthetic agents. If the organism is killed or inhibited by the concentration of the antibiotic.

Swab a Mueller-Hinton plate with each of the bacteria. Swab the surface of the agar completely (you do not want to leave any unswabbed agar areas at all). 3. . In the pictures above and below. and result reported as S (sensitive). If you do not see 8 disks come out onto your agar plate.Page 92 THE PROCEDURES: 1. Just quickly pick up the disc and move it to the appropriate place with the sterile forceps. R (resistant). Dip a sterile swab into the broth and express any excess moisture by pressing the swab against the side of the tube. report it as 0---even though the disc itself is around 7 mm. o Lightly touch each disc with your sterile inoculating loop to make sure that it is in good contact with the agar surface. o Each free antibiotic cartridge should have a little metal arm that allows you to dispense the disc right onto the agar. Record the results for everyone on your table in the table below. looked up on the lab chart. Even so. INTERPRETATION: 1. at the widest diameter. you will have to manually remove the antibiotic from a free cartridge (see line below). 3. you can see what happens when the plate is not swabbed correctly with even coverage of the bacterium over the entire agar. Place the metric ruler across the zone of inhibition. turn it 90 degrees and repeat the swabbing process. or I (intermediate). The disc diameter will actually be part of that number. Incubate upside down and incubate at 37o C. 2. 5. sometimes the discs pop out and fall in a place on the agar that you do not want it to be. If there is NO zone at all. HOLDING THE PLATE UP TO THE LIGHT MIGHT HELP. 4.) Run the swab around the circumference of the plate before discarding it in the discard bag. THE ANTIBIOTIC DISKS: o The antibiotic dispensers have 8 antibiotic cartridges in them. Allow the surface to dry for about 5 minutes before placing antibiotic disks on the agar. 5. After completely swabbing the plate. (It is not necessary to re-moisten the swab. 2. and measure from one edge of the zone to the other edge. Zone diameter is reported in millimeters. 4.

Methicillin and ampicillin are semisynthetic drugs. S. S. Record the results for the 5 bacteria with all of the antibiotics. or I? zone diameter in millimeter units Use chart for sensitive. S. R. The larger the zone size. or I? Bacillus Zone Dia. the more ____________ the bacterium is to that antibiotic. R. or I? Staph Zone Dia. coli Zone Dia. What class are these drugs in? What does semisynthetic mean? 5.Page 93 LABORATORY REPORT SHEET QUESTIONS: 1. or I? Pseudomonas Zone Dia. or intermediate 2. or I? E. What measurement units are used to measure the zone sizes? 4. S. resistant. aerugenosa compare in its sensitivity to the other four bacteria? . How does Ps. 3. R. R. ANTIBIOTICS Strep Zone Dia. R. S.

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hands. between the fingers. while Povodone is an iodophor. you will also learn about the tenacity of your normal flora and how difficult it is to rid yourself of them. Resident normal flora are deeply entrenched in ecological niches. • Wash each side of the arm to three inches above the elbow for one minute. • Scrub each side of each finger. the scrub must be lengthened by one minute for the area that has been contaminated. iodine compounds and iodophors. not the procedure listed above. chlorhexidine. keeping the hand higher than the arm at all times. a combination of iodine and a surface-active agent that frees elemental iodine in solution. Do not move the arm back and forth through the water. Accepted antimicrobial agents include alcohols. MATERIALS NEEDED: per table 6 BHI (brain heart infusion) agar plates (3 for left hand/chemical. • Clean areas under the nail with a nail file. In addition to testing a couple of antiseptic chemicals. 3 for right hand/handsoap) chloroxylenol (Ultradex/PCMX) OR an iodine (Povodone Iodine) surgical scrub brush 1 regular scrub brush Pump handsoap will be at the sinks sterile paper towels . stick. Understand the standard scrub procedure. keeping hands above elbows at all times. phenolics. and quaternary ammonium compounds • Scrub arms. or brush. and forearms • Reduce the resident microbial count to a minimum. If the hand touches anything except the brush at any time. Triclosan. and the back and front of the hand with the antiseptic for 3-5 minutes. both bacteria (Staphylococcus. and • Inhibit rapid rebound growth of microorganisms Page 95 The standard procedure (from INFECTION CONTROL TODAY online) • Wash hands and arms with antimicrobial soap. Micrococcus. traded between people when shaking hands or when you open the door of the bathroom. There are a variety of microorganisms on the skin. The transient normal flora come and go. • Repeat the process on the other hand and arm. OBJECTIVES: Compare the killing power of different aseptic chemicals. from fingertips to elbow.THE SURGICAL HANDSCRUB The purposes of a surgical handscrub (even though hands are protected by gloves) • Remove debris and transient microorganisms from the nails. Corynebacterium rods) and fungi (yeasts such as Pitysporum). • Rinse hands and arms by passing them through the water in one direction only. Chloroxylenol (also called PCMX) is a halogenated phenolic compound. You will be using a modified procedure for this lab. such as pores and ducts.

1. and the ASSISTANT will dry that hand. and “C”. but one set will be LEFT hand (iodine or chloroxylenol) and the other set will be RIGHT hand (handsoap). At the sink. for 15 seconds. Both sets will be labeled “A”. 6.50 colonies = light growth = +1 less than 5 colonies = sterile (practically)= – chemical Left hand Right hand plate A plate B Which chemical used? plate C . FIRST. The ASSISTANT will use a sterile paper towel to dry the left hand of the SURGEON. up to the wrist (NO hand soap). 3. The ASSISTANT will now use the iodine or chloroxylenol scrub brush over the entire left hand of the SURGEON for 2 minutes. “B”. • A plates – before anything done to the hand • B plates – after using a scrub brush with water for 15 seconds • C plates – after using the designated chemical with scrub brush for 2 minutes 2. 9. the ASSISTANT will thoroughly scrub the left hand of the SURGEON with water ONLY. RESULTS Record results as follows: more than 50 colonies = heavy growth = +2 5. The SURGEON will rinse the left hand in tap water. Do not touch anything else! 7. Do not touch anything else! 4. Incubate all BHI agar plates at room temperature.Page 96 THE PROCEDURE: One person on the table is being scrubbed. henceforth called the “surgeon. Use the 3 middle fingers on the left hand to press onto plate “LEFT B”. Make a control plate---no action performed on the hands.” The “assistant” will help with the scrubbing and drying of hands. The ASSISTANT will rinse the SURGEON’s left hand in tap water. This will be the “LEFT A” plate. Use the 3 middle fingers on the left hand to press onto plate “LEFT C”. label the 2 sets of BHI agar plates. the same thing will be done in steps 2-7 with the RIGHT hand of the SURGEON. 8. rather than use a chemical you will be using a pump handsoap. 5. NOW. Use your middle 3 fingers on your left hand to press onto the BHI plate. And in step 5.

Which normal flora---residents or transients---were the first to be killed during the first scrub? 2.Page 97 LABORATORY REPORT SHEET QUESTIONS: 1. Are normal hands washings with antimicrobial soap effective to remove microbes? . Can one easily get rid of resident normal flora? 3.

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called a plaque. The mix is incubated in the water bath. The procedure is really very easy.Page 99 BACTERIOPHAGES Bacteriophages are viruses which infect bacteria. Most bacteria have phages that are able to parasitize them. The purpose of using 2 different viruses is to show the specificity of a virus for its host. known as phage typing . and generally refers to a virus. In this lab. so these bacteriophages are called coliphages. the ability to be infected with a known phage type is used to identify some strains of bacteria (like Staph). As the surrounding cells are infected and killed by the released viruses. • • • The phage specimen is diluted. 2 kinds of bacteriophages will be used---T4 and phi 174 viruses. After incubation the mix is added to the soft agar and poured over the tryptone agar plates. The plaques can be counted and the number of virus particles or virions in the original specimen. PHAGE (as in phagocytosis) means "to eat". even for these little bacterial viruses. Identify viral plaques in a bacterial lawn. As the virus infects bacterial cells that it has been mixed with. MATERIALS NEEDED: per table 10-3 dilution of the bacteriophage 1ml pipettes and pi-pump 5 . Their host bacteria are 2 different strains of E. OBJECTIVES: Learn how to culture viruses in a host cell. Quantitate viruses in an specimen.9ml saline for dilutions of bacteriophages 50oC water bath 1 strain of E. The bacteria have been poured into what is called a bacterial lawn on the agar plate. a clear spot on the agar---in the bacterial lawn--develops. In fact. Bacteria and phage are mixed together in tubes of soft agar. coli (B or C)in TSB and 1 type of virus (T4 and phi 174) 6 TSA plates 6 . can be quantitated as viruses/ml of plaque-forming units/ml. the lytic infection destroys the bacteria. coli.4 ml soft agar tubes (kept in water bath the entire time) .

1ml of each dilution into 1 soft agar. 4. 6. Starting with the 10-3 dilution you received. • • T4 and E. Remove 1 soft agar at a time and pour it onto the TSA agar plates. transfer 1ml to the dilution tube marked 10-4 and mix. and inoculate 0. Make dilutions of the virus 2. gently rotating the plate WELL so as to distribute the phage-bacteria all over the agar. Although you may not have the ability use a sterilizing burner. Keep these soft agars in the water bath to keep from solidifying. You will be making 1/10 dilutions. Set up 5 saline (0. Each table will use a different combination of a phage and an E. and transfer 0. 10-6. Mix these well. coli B phi 174 and E. Allow the plates to harden and incubate at 37oC right side up. 7. 10-5. . 5. 3. coli C 1. Pour the soft agar (virus + bacterium) onto pre-made agar plates. use your BEST aseptic technique. Make 4 more dilutions out to 10-8. coli to the soft agar. Take your E. coli-soft agar mix. 3. Be SURE to mix the dilutions well.3ml into 6 (LABELLED 10-3 to 10-8 ) soft agar tubes. Now take your 6 viral dilutions (10-3 to 10-8) in a rack over to the water bath. coli host. Change pipette s between dilutions. Add E.Page 100 THE PROCEDURE: The 4 parts of this exercise: 1.85% NaCl) dilution tubes labeled 10-4. 4. 2. Add the viral dilutions to the E. 10-7. coli over to the water bath. and 10 -8.

# viruses/ml = P F U s dilution of tube X amount plated 4. Get an accurate count of that plate. and determine which one has between 30300 plaques (you can also use the Quebec colony counters---good backlighting!) 3. . hold it up to the light. of original specimen. Pick each plate up.INTERPRETATION Page 101 1. from lowest dilution towards highest dilution. Calculate the number of viruses per ml. 2. Lay the 6 plates right side up. Fill in the formula for viral counts.

coli added to the soft agar overlays? 3. Give the plaque count/ml for your viral specimen. . Why did the two phages not grow on both E.LABORATORY REPORT SHEET QUESTIONS: 1. coli strains? 4. Why are viral counts expressed as plaque forming units (pfu)? Page 102 2. Why was E.

you will be able to fall back to your reserve stock. Determine physical characteristics of the organism provided 3. along with the given reference flow chart and identification texts. It is critically important that aseptic techniques are used during transfers and inoculations to prevent contamination of your cultures. 5. Working stock cultures will be used to inoculate the various biochemical test media over the next several weeks and should be fresh and free from contaminants. Maintain a pure culture of the unknown organism provided 2. whether positive or negative. OBJECTIVES: 1. The instructor will not provide a new culture for you to start with in the middle of the unknown exercises. biochemical testing has been used to make bacterial identification down to the “species” level. These schemes are based on creating and matching biochemical profiles of the production of enzymes. Identification schemes and flow charts can be found in reference texts such as “Bergey’s Manual of Determinative Bacteriology” or “The Prokaryotes”. kept with the original slant in the refrigerator. Inoculate various biochemical tests and be able to read and understand the significance of each test. Use the information generated by testing.IDENTIFICATION OF UNKNOWN BACTERIA Page 103 It is virtually impossible to identify bacteria based on physical characteristics alone. to deduce the Genus and Species designation of the unknown organism provided. If contamination is suspected. Hand in a report of the testing performed (Unknown identification sheet). Each student will hand in their own separate report even though you have performed the work together as a group. A reserve stock culture should be made and after incubation and comparison with the original slant. Instead. If you fail to maintain a reserve stock you will not be able to recover your organism if disaster strikes. This is due to the fact that there are only a few basic shapes and physical features commonly seen in the prokaryotic world. MATERIALS NEEDED: TSA plates TSA slants TSB broths Clean glass slides Gram stain reagents Oxidase strips and reagent . acids and gases by isolated pure cultures of a given microorganism. 4. Each group of students will receive a pure culture of an unknown bacterium belonging to the Family Enterobacteriaceae. It is the responsibility of the group to maintain stock cultures of the organism provided. Remember it is import to keep your own journal and not to plagiarize other students in the group. a journal of how you arrived at the identification you indicated and a TSA plate containing the unknown organism streaked to demonstrate isolated colonies.

These tests are the HIGHEST PRIORITY TEST results. Gram stain your unknown organism using the TSB broth culture. catalase & oxidase tests start biochemical tests more biochemical tests more biochemical tests 2 streak plates---1 to turn in for grade. indole reaction and motility. Nitrate broth SIM tube (H2S. Inoculate a TSB broth from the original slant 3. the test reactions most important in the identification of your organism. 3. 7. Place the original stock slant labeled with your group names and instructor in the refrigerator. Prepare a new working stock TSA slant from the previous working stock. TSA plate. Observe the biochemical reactions and record the results on your unknown identification sheet. texture. 6. Make any additional notes in your journal. Run the oxidase and catalase tests (exercises follow) 5. 3rd Session 1. Incubate all cultures at 25 c or 37 C as directed by your instructor. In order to confirm the oxygen requirements of your unknown you will perform the oxygen requirements exercise using the known organisms provided and your unknown organism. 5. Observe your new slants looking carefully for signs of contamination. 2. Describe the colony morphology displayed by isolated colonies observed on the streaked plate (refer back to the Colony Morphology experiment). other for API20 test next period run API20 finish reading all test. see IMViC tests) Simmons Citrate agar (see IMViC tests) . Note the shape. using the original agar slant culture. Streak a TSA plate for isolated colonies using the 3 section method. opacity and odor. prepare to turn in unknown identification 1st Session 1. 2. arrangement and Gram reaction of your organism. do gram stain run oxygen requirements. Compare to the original slant noting the color (note pigmentation). Inoculate the first series of biochemical tests using your working TSA slant culture. 2nd Session 1. (see Oxygen Requirements exercise). 2. 4. Record the oxygen requirements of your unknown organism using the reference organisms as a guide. 4. 6.Page 104 Various biochemical media (distributed in sets during subsequent lab periods) THE PROCEDURES: SEQUENCE OF EVENTS FOR UNKNOWN IDENTIFICATION 1st session 2nd session 3rd session 4th session 5th session 6th session 7th session 8th session Get unknown inoculate TSA slant. colony morphology. Incubate all cultures as directed by your instructor. Label two slants (one for reserve and the other as the working stock) and inoculate using an inoculating from the original culture you have been given.

Hand in your Unknown identification sheet (each student). 2. Prepare a fresh working stock TSA slant in case any additional tests are required next day to finalize identification of the unknown.mannitol . 4. Use your fresh working stock slant to inoculate the next series of biochemical tests: 3. Use your fresh working TSA slant to inoculate the next set of biochemical tests Hydrolytic tests (refer to the section on each specific test in your lab manual) Starch plate Casein plate Lipid plate (same as lecithinase test) Urea broth Gelatin broth 4. Read and record final tests results according to each separate procedure in the laboratory manual. If you are still unsure of the identity of your unknown. inoculate other tests as indicated in the reference texts for next class. 2. Observe the biochemical reactions and record the results on your unknown identification sheet. Make any additional notes in your journal. One plate is to be used to inoculate a biochemical test strip called an API20e.glucose .sucrose 6th Session 1. 5th Session 1. Review the resources available in the laboratory (Bergey’s Manual and other reference textbooks). A second plate should contain the organism streaked to show isolated colonies and will be handed in with the reports. Incubate all cultures as directed by your instructor. 2. your journal (each student) and the streaked plate of the unknown (1 per group) . Do they match? If they don’t you will have to try to make the best match that you can with the information you have collected. 4th Session 1. Use your fresh working TSA slant to inoculate the next set of biochemical tests Oxidation-fermentation tests: (read sections on each specific tests first) Phenol red sugar broths with Durham tubes (gas production) . Observe the biochemical reactions and record the results on your unknown identification sheet. Streak fresh TSA plates for isolated colonies.lactose . Double check your own results with the identification made using the API20e test strip. 7th session Inoculate the API20e as described in the appropriate exercise.Page 105 Phenylalanine deaminase agar (Decarboxylation & Deamination of Amino acids) Decarbolyxase broths (Decarboxylation and Deamination of Amino acids) 3. Make any additional notes in your journal. Prepare a new working TSA slant for the next day. 8th Session 1. 2.

Page 106 LABORATORY REPORT SHEET QUESTIONS: 1. What other methods are available for identification of bacteria to the species level? 3. Are there methods for identifying bacteria below the species level? What are some examples of this and how useful are they? 4. purify and identify specific bacteria? . Why is biochemical testing used to identify bacteria to the species level? 2. Why is it important to be able to isolate.

BACTERIA Aerobe Facultative anaerobe Page 107 oxidase + motility These are key tests for ID. For the key to work for you.GRAM . be sure that you are accurately interpreting the test. but NOT the only test differences among organisms. oxidase - + indoie H2S - + Nitrate reduction + Chromobacterium Aeromonas + Phenylalanine deaminase Phenylalanine deaminase Burkholderia Acinetobacter + VP + + - citrate Proteus citrate Some sugars used motility Escherichia + + Morganella Providencia lysine + ornithine + - motile nonmotile Pseudomonas Branhamella Klebsiella + DNAse Providencia Citrobacter + Alcaligenes Neisseria + Salmonella Serratia Enterobacter . This lists the predominant test reaction (there may be a species which is the opposite reaction.

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Microscopy is the most accurate way to determine motility. Some cells will "run" straight across the field. Second. NOTE: Strict aerobes may not grow well or at all in this medium. occuring as a result of bacterial metabolism. Another way to determine motility---TTC motility agar with tetrazolium---will be used in lab. Motile bacteria move about with structures called flagella (a few exceptional bacteria move with the help of axial filaments. First of all. and 2) use a young culture of bacteria. The keys to a good hanging drop slide are 1) use a small drop of bacterial suspension.the hanging drop wet mount . and they are small: therefore. even with the oil immersion lens. Nonmotile bacteria without flagella are called atrichous.motility agar media (SIM and tetrazolium motility agars used later) Looking at living bacteria are not as easy as one would think. Kinetic energy inherent to all molecules causes this kind of movement. but do not let it dry out. others will "tumble" across the field in a slower motion. even nonmotile ones. living bacteria have no color. On the other hand. those bacteria with flagella will be very apparently moving about the field of vision. . Categories of flagellation: • monotrichous = single flagellum • peritrichous = flagella all around • amphitrichous = flagella at both ends • lophotrichous = tuft of many flagella at one end or both ends Motility can be identified in a couple of different ways: . Not only can motility be identified. assuming that you have a fresh culture of bacteria.MOTILITY TESTS Page 109 There are a variety of ways to determine motility of a bacterium—biochemical tests as well as microscopic analysis. but also the organization and number of flagella. The tetrazolium is a colorless salt which becomes red when reduced. they are really difficult to see. Identify flagella on bacterial cells. This Brownian movement is caused by water molecules bouncing around in the solution. knocking up against each other and the microorganisms. which cannot be seen in the microscope). Differentiate among different types of flagellation. Identify motility using different methods. The tetrazolium makes the motility agar much easier to read for motility. all bacteria have some vibrational movement. OBJECTIVES: Differentiate between Brownian movement and true motility. although perhaps not all of the bacteria will be moving.

Place a drop of the bacterial culture (optimally from a young broth culture) in the middle of a cover slip. c. It should look like the diagram below. wet mount/hanging drop a. E. The jelly holds the cover slip to the slide and also keeps the suspension from drying out. Be sure to remove the oil with the lens paper. • You will have to move around the slide to find the best field of vision. BUT with the added.Page 110 MATERIALS NEEDED: per table prepared flagella stains (mixed flagella slides . prepared flagella stains • These stains are bought and ready to use. If NONMOTILE. Incubate at 30 or 37 degrees C. 2.amphitrichous. and Pseudomonas (less than 24 hours old optimally) 2 Motility agar deeps with tetrazolium dye cover slips depression microscope slides vasoline jelly in syringes THE PROCEDURES: all procedures done as a table Microscopy 1. you will see the intact straight stab line. helpful colored dye tetrazolium which turns red as a result of the bacteria metabolizing. 2. all the way to the bottom. the original stab line will diffuse out into the medium as the bacteria spread throughout. atrichous. you still use oil when on 100X magnification. Hold the tube up to the light and look at the stab line to determine motility. the flagella will break off and you may not see many in some areas of the slide. Inoculate E. 3. Makes 2 hanging drop slides---Pseudomonas and Klebsiella. Often. . surrounding line of petroleum jelly all around the edge of the cover slide. Turn the depression slide upside-down (depressed area facing down) and gently touch the cover slide. b. Although they have cover slips. Biochemical media 1. d. Place a thin.peritrichous) fresh TSB cultures of Klebsiella. Now flip the entire microscope slide/cover slip combination over.coli and Staph cultures into tubes of motility agar with tetrazolium dye with a NEEDLE. Proteus slides . If MOTILE. coli. Look for the spread of the inoculum away from the inoculation line.

What feature of the bacterial culture will increase the probability of true motility? ..Page 111 LABORATORY REPORT SHEET QUESTIONS: 1. How do you tell if the organism is motile? 6. peritrichous Proteus amphitrichous bacterium 2. What designation does one used for a bacterium without flagella? 4. What is the function of tetrazolium? 5. Make drawings from the 2 prepared slides. Draw the 2 tubes of TTC media with the growth patterns of the 2 bacteria. What is the cause of Brownian movement? 8. Why is petroleum jelly used to make this slide? 7. Staph Pseudomonas 3.

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The candle jar at right has 3-5% CO2 and 8-10% O2 (0. The paper sachet contains ascorbic acid and activated carbon which reacts on exposure to air. and reduced oxygen at less than 10% (candle jar)--and compare the quantity of growth. Because a GasPak jar looks the same. The Gram + genus Bacillus and Gram – genus Pseudomonas include aerobic bacillus-shaped bacteria. whereas some will not grow at all (e. When the paper sachet is placed in a sealed plastic pouch. is included in the jar. Some microaerophiles are actually capnophilic (requiring elevated CO2 levels to grow). with a gas generator envelope inside. Oxygen is rapidly absorbed and CO2 is produced. On the left is a GasPak jar. By studying growth in different environments. superoxide radicals and hydrogen peroxide. The environment is 0% O2. Neisseria gonorrhoea). since they grow optimally under reduced (but present) oxygen conditions as in the candle jar. whether it has oxygen inside or not. Possibly the by-products of aerobic respiration. Strict aerobes may not grow well in a candle jar. no oxygen at all (GasPak jar). producing 10-15% CO2. containing methylene. one can determine whether the organism is a facultative anaerobe. Many microaerophilic bacteria will grow poorly at 22% O2.. make it difficult for the microaerophiles to do well in 22% O2. This is a handy way to determine if you have an aerobe which is microaerophilic. colorless when reduced. The carbon within the puch reacts with free oxygen in the jar. this reaction will create ideal atmospheric conditions for the growth of anaerobes. Methylene blue is blue when oxidized. depending on the species. an anaerobe.g.OXYGEN REQUIREMENTS & CULTURING ANAEROBIC BACTERIA An excellent way to determine the oxygen needs of a bacterium is to grow it in different oxygen environments---atmospheric oxygen of 22%. an aerobe or a microaerophile. respectively). an indicator strip.3% and 21% in the atmosphere. . Page 113 The newer anaerobic system (seen at left) consists of a plastic container (for the agar plates) and a paper gas generating sachet.

OBJECTIVES: Identify the 3 major categories of microbes based on oxygen requirements. and Gram + Clostridium (tetani. exemplified by the bacillus-shaped genera---Gram – Bacteroides. some oxygen will be in the tube between the cap and the broth and there is no way to get rid of it. All of our incubators are ambient air incubators: O2 comes in from the outside atmosphere. However. So there will be some diffusion of oxygen into the top portion of the broth. but they do not use it. and that is where any aerobic bacteria may grow. • Ambient air is 22% O2. the methylene blue strip should be checked to make sure that it is COLORLESS (verifies anaerobiosis). An indicator. are the facultative anaerobes which prefer to use O2 when present but will grow without it. Learn different ways to culture anaerobic bacteria. boil the broth for 5 minutes (removes the oxygen). growth is indicated by gray area CAUTIONARY NOTES: • Do not shake the thioglycollate broth. . Oxygen will permeate the broth then this medium sits around for a while. facultative anaerobe. Aerotolerants are anaerobes that can grow in the presence of O2 (compared to the strict anaerobes which would likely die). Check for the pink color: if so. Where the bacterial growth is located in the tube indicates whether it is an anaerobe. to reduce oxygen entry. botulinum). Another way to culture and grow anaerobes is the use of reduced media---media without oxygen. resazurin. • Before the anaerobic jar is opened.Page 114 Quite a few human pathogens are strict anaerobes. Thioglycollate broth has a reducing agent in it---the chemical thioglycollate---which binds any free oxygen within the medium. You will also notice that these tubes have screw caps. in the medium will be a light pink in the area of higher oxygen. allowing a tight closure. or an aerobe. Identify the oxygen requirements of your unknown bacterium. but very common. Bacillus (anthracis). And last.

or +2 (good growth).ambient air and GasPak anaerobic jar. Inoculate the section by streaking a straight line or a a slight zig-zag. +1 (slight growth). • Record your results in the LAB REPORT SHEET. .Page 115 MATERIALS NEEDED: 1 thioglycollate broth per bacterium – 4 total 2 TSA plates (divide into 4 sections) GasPak jar for entire lab GasPak envelope for the jar methylene blue indicator strip for the jar cultures: Clostridium species. AND your unknown THE PROCEDURES: per table Thioglycollate broth 1. 2. Place your plates. 3. coli. • Record the results of your unknown bacterium in your journal. anaerobic. • Record the results of your unknown bacterium in your journal. You will use 1 thioglycollate broth for each organism. or facultatively anaerobic. The thioglycollate broth should be either boiled first before inoculation OR recently made so that the oxygen content is very low. the bottom. top to bottom. • Determine whether each organism is aerobic. Divide the plates into sections. upside-down. in the correct location---ambient air 37C incubator or GasPak anaerobic jar. E. Your instructor will make sure that the jar has a methylene blue indicator strip inside. be sure that you inoculate all plates using the same technique. Label 2 plates for the table--. as well as the quantity of growth on the 2 plates. 3. 4. He or she will also place the jar in the 37 C incubators INTERPRETATION: after incubation TSA plates • Compare the presence/absence of growth. one for each bacterium. HOWEVER. Inoculate a tube of thioglycollate broth: make sure that the loop or needle goes down to the BOTTOM of the broth (do not get metal holder in the sterile broth). 4. Thioglycollate broth • Determine WHERE the most amount of growth occurs in the column of liquid---the top. 2. and Micrococcus luteus. DO NOT SHAKE IT! • Record your results in the LAB REPORT SHEET. Incubate at 37 degrees C TSA plates in 2 different oxygen environments 1. Your instructor will tell you what to do. 5. Quantify the growth as – (no growth).

Differentiate between an aerotolerant and a facultative anaerobe. Record as -. coli Clostridium 6. Data from thioglycollate broths. 5. +1. or +2 growth. Shade in where the growth is located for the 3 bacteria. 4. Data from TSA plates in different environments. Why should you boil thioglycollate broth if it is not freshly made? 2. or C—is the strict anaerobe? . Why might one use a candle jar for incubation of a bacterium? 3. B. Bacillus E.Page 116 LABORATORY REPORT SHEET QUESTIONS: 1. Which of the following bacteria---A.

CARBOHYDRATE UTILIZATION

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Sugars are very important tests for a lot of microbes. Unfortunately, there are a few problems related to sugars. Whereas most biochemical media is stable, allowing you to inoculate one day and read a couple of days later, sugar is NOT. The problem is that there are other nutrients in the media that can be used by the bacteria, like proteins. Although the sugar is the primary nutrient used (if the organism uses that particular sugar), when the microbe runs out of it, protein or other nutrients will be attacked. This can cause changes in the color of the medium because there is a pH indicator added to detect acid production. When proteins are used, alkaline by-products are produced and the medium can change colors. If you let these sugar tests go for more than a day, you risk the possibility that the color will have changed and you may call the test result negative rather than positive. Therefore, it is a good idea to run into the lab and read these sugar tests somewhere between 8-12 hours, if at all possible. There are no reagents that have to be added since the pH indicator is added to the medium already. You are going to see 2 different ways to run sugar tests: phenol red sugar broths and the sugar disc methods. Some of the sugars come in phenol red broth, already with sugar in it lactose, glucose (dextrose), and sucrose. Or, there are a few sugars that do not come as a disc form nor do we have pre-made phenol red broth with the sugar. For the sugars maltose, arabinose, and some others, we will add the sugar to the phenol broth as asked for. Please just ask if you need either of these sugars OBJECTIVES: Learn different test procedures for determining carbohydrate use. Identify different end products of sugar use. MATERIALS NEEDED: phenol red sugar broths (lactose, sucrose, glucose/dextrose) TSB small sterile tubes 1ml pipettes pi-pump sugar discs ethanol forceps THE PROCEDURES:
for PHENOL RED BROTH: The broth can have various sugars added to it. It has a small upside

down tube called a Durham tube that collects CO2 gas. 1. Inoculate the phenol red glucose broth with your unknown bacteria. 2. Inoculate the organism into the tube with the disc and incubate at 25 or 37 degrees C.

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for SUGAR DISCS: You will need some forceps and the ethanol to maintain sterility of the sugar

discs when transferring them. There are at least a couple of dozen sugars that we have in the refrigerator, but today you are using sucrose. 1. Obtain a tube of TSB broth and aliquot 0.5ml into a sterile tube. 2. Sterilely transfer a sugar disc into the new tube (using either STERILE forceps dipped in alcohol and flamed OR dispensed with the metal holder on the sugar cartridge). 3. Inoculate the organism into the tube with the disc and incubate at 30 or 37 degrees C. 4. Record the results of your bacterial unknown in your journal. If you do not find the sugar that you need for identification, PLEASE ASK.

INTERPRETATION:
PHENOL RED: You are looking for a change in the phenol red indicator to yellow, for acid (A)

production. In addition, there may be CO2 gas (G). The results can be reported as A/G, A/-, /G, or -/-. You may notice an alkaline change to a very red color, indicating basic end products, but we don't care about basic reactions. As above, you are looking for the yellow acid color. There is no way to determine gas production with this method.
SUGAR DISCS:

BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB.

LABORATORY REPORT SHEET
QUESTIONS: 1. The pH indicator used in most sugars is:

2. Why should sugars always be read within 12 hours, IF AT ALL POSSIBLE?

3. What is the purpose of the durhm tube?

4. Sugar, when catabolized, is broken down to:

CASEIN HYDROLYSIS

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The enzyme caseinase is secreted out of the cells (an exoenzyme) into the surrounding media, catalyzing the breakdown of milk protein, called casein, into small peptides and individual amino acids which are then taken up by the organism for energy use or as building material. The hydrolysis reaction causes the milk agar, normally the opacity of real milk, to clear around the growth area as the casein protein is converted into soluble and transparent end products—small chains of amino acids, dipeptides, and polypeptides. OBJECTIVES: Identify the reactions associated with growth on skim milk agar. MATERIALS NEEDED: 1 skim milk agar plate per table THE PROCEDURE: 1. 2. 3. 4. Run this test using your unknown bacterium. Inoculate the organism on the plate either a straight line or a zig-zag. Incubate at 25 or 37 degrees C. Record the results of your bacterial unknown in your journal.

INTERPRETATION: Hold the plate up to the light to see the zones. Positive reactions may be recorded as strong + or weak + reactions. There is no reagent or indicator in the agar. A zone of clearing around the growth area identifies the presence of the enzyme caseinase.

BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB.

3.Page 120 LABORATORY REPORT SHEET QUESTIONS: 1. What is the difference in a weak + and a strong + reaction on the agar plate? 2. What is an exoenzyme? . The substrate for this enzyme is _____.

but it may kill the culture. MATERIALS NEEDED: 3% hydrogen peroxide THE PROCEDURE: 1. OBJECTIVES: Test for the enzyme catalase on your unknown isolates. INTERPRETATION: Usually immediately you will see a reaction if there is one. Catalase is an enzyme that can degrade the hydrogen peroxide in the cell before it can do any cell damage. Record the results of your bacterial unknown in your journal. Add one drop of H2O2 and record results. 3. It splits the H2O2 to free oxygen (bubbles) and water. Hydrogen peroxide is a by-product of respiration and is lethal if it accumulates in the cell. . IMPORTANT POINTS: • • Use a culture growing on an agar plate or agar slant to run the slide catalase test. You can drop H2O2 directly on an agar plate or slant. Slight bubbles indicate a positive reaction also.CATALASE TEST Page 121 Catalase is an enzyme that splits hydrogen peroxide into water and oxygen. Pick the inoculum from a plate culture or slant culture and place it on a slide. 2. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. most often lots of bubbles.

Name the reagent used for this test.Page 122 LABORATORY REPORT SHEET QUESTIONS: 1. . What are the bubbles that you see in a + reaction? 2.

5. . BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS. MATERIALS NEEDED: 1 DNAse agar per table 1 N Hydrochloric acid culture of Staphylococcus aureus THE PROCEDURE: 1. there will be clear zones seen. Divide the DNAse agar plate into 2 parts. 4. but HCl reagent is used to precipitate out the undestroyed DNA. 2. you will notice a light tan zone around the growth area. Deoxyribonuclease is an enzyme excreted from the cell which will break the DNA down into smaller molecules. Staphylococcus aureus. The lab may be using DNAse with methyl green indicator instead of regular DNAse agar. Incubate at 25 or 37 degrees C. Record the results of your bacterial unknown in your journal. Around the growth area. The rest of the plate with the remaining DNA will have turned an opaque white. and the other side will have the + control. AFTER INCUBATION: Flood the plate with HCl and wait for 1 minute. If using the DNAse with methyl green. HCl. On 1 side you will inoculate your unknown organism. OBJECTIVES: Identify the presence of DNAse enzyme. INTERPRETATION: The interpretation of the reaction requires a reagent added to the culture after it grows. There is no indicator in the medium. if the bacterium has made DNAse and destroyed the DNA. 3. Within a minute after addition of the reagent. NOTE.DNASE PRODUCTION Page 123 Deoxyribonuclease (DNAse) agar actually has DNA in it. The indicator methyl green is used rather than the HCL reagent. Inoculate one DNAse agar plate with the unknown organism---a straight line inoculation. a clear zone (large or small is +) around the growth area. BE SURE THAT THE PLATE IS SITTING ON THE BLACK TABLE TOP: The black will enhance the zone identification.

What is the purpose of the methyl green in this medium? .Page 124 LABORATORY REPORT SHEET QUESTIONS: 1. Why look at the DNAse agar against a black background? 3. When HCl is added to this agar. what happens to remaining/uninoculated DNA in the agar? 2.

plus I have added a 3rd that you may want to run at a later time. Since the medium has sugar in it. A base broth without amino acid is run on each organism as an inoculated control. and most of them can be used by one bacterium or another. This is NOT a reaction reaction that you record. In this lab you will look at 2 different amino acid tests. no amino acid added Moeller ornithine decarboxylase broth Moeller arginine decarboxylase broth Moeller lysine decarboxylase broth phenylalanine agar slants mineral oil pipette and pi-pump AFTER INCUBATION: FeCl3 reagent . There are 3 decarboxylase enzymes we can test for---arginine decarboxylase. the end product is a basic chemical which causes the pH to go up. We run one deaminase test---phenylalanine deaminase---which uses FeCl3 as the reagent. The deaminases do the opposite. and lysine decarboxylase. As a result.DECARBOXYLATION & DEAMINATION OF Amino Acids Page 125 These are about 20 amino acids. changing the indicator brom cresol purple to turn purple. NOTE: The decarboxylase test needs to be anaerobic (assuming your unknown is NOT a strict aerobe). OBJECTIVES: Learn the various methods for determining deamination and decarboxylation. reacting with the phenylpyruvic acid that results from the breakdown of phenylalanine. and producing chemicals which are acidic. turning the indicator yellow. MATERIALS NEEDED: Moeller base broth. you are making sure that the organism uses the sugar. ornithine decarboxylase. so you overlay the broths with a layer of sterile mineral oil. These enzymes break the bond holding the carboxylic (COOH) group to the rest of the amino acid. knocking off the amino groups. Many of the biochemical tests are based on protein and amino acid use.

Page 126 THE PROCEDURE: 1. no reagent added (it already contains a ph indicator. 5. . LYSINE. Record the results of your bacterial unknown in your journal. 2. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. Incubate at organism's optimal temperature. o 6-8 drops of ferric chloride are added to the phenylalanine agar slant. as a result of the decarboxylation process. turning the slant an avocado green. the bromcresol purple pH indicator will be a purple or purple-gray (yellow is acid. ORNITHINE DECARBOXYLASE BROTHS: In a basic pH. 4. INTERPRETATION: ARGININE. 3. 25 or 37 degrees C. Inoculate the phenylalanine deaminase slant as you would a normal slant. but is a negative reaction). The layer should be 1/4-1/2 inch deep. for a couple of days. AFTER INCUBATION: o The decarboxylase broths are read as is. Bromcresol purple). washing down the slant. PHENYLALANINE DEAMINASE: The FeCl3 reacts with the acid produced as a result of deamination. Inoculate the decarboxylase broth (along with a base without amino acid) with the unknown bacterium and overlay with a layer of mineral oil (NOT immersion oil).

What is the amino acid in the deamination agar? 4. 2. In the presence of the enzyme decarboxylase. Why add mineral oil to the decaroxylase amino acid broths? 3. the amine side chain of the amino acid molecule will be cleaved off---TRUE or FALSE?.Page 127 LABORATORY REPORT SHEET QUESTIONS: 1. Name the pH indicator in the Arginine decarboxylase test – Phenylalanine deaminase test - .

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GELATIN HYDROLYSIS

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Gelatin is a protein that is solid at room temperature. If the bacterium makes the enzyme gelatinase (which optimally is produced at 25C, not 37C), the gelatin is hydrolyzed and becomes a liquid. There is no indicator or reagent added, simply solidification or liquefaction. OBJECTIVES: Learn the method for determining gelatin hydrolysis and the reactions produced. MATERIALS NEEDED: 1 nutrient gelatin deep for unknown THE PROCEDURE: 1. Stab the gelatin deep with a needle all the way to the bottom (being careful NOT to get the metal holder into the agar). 2. Incubate at 25°C for a couple days, up to a week. Keep the medium for a week if negative. 3. Record the results of your bacterial unknown in your journal. INTERPRETATION: The tube is placed on ice for about 15 minutes, or in the fridge for about 30 minutes, to determine liquefaction. If the tube has been incubated at 37C it will be liquid when taken out of the incubator, so remember to place it on ice before calling the reaction. The liquefaction can be complete throughout the tube or perhaps partial. If negative, keep the medium at 25C for a week to make a final call of negative. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB.

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LABORATORY REPORT SHEET
QUESTIONS: 1. Why place this medium on ice to test for a positive reaction?

2. This gelatinase enzyme is temperature-sensitive, and preferably works best at what temperature?

The citrate test identifies the use of citrate as a sole carbon source. perhaps. The basic end products will cause the brom thymol blue indicator in the medium to turn from forest green to royal blue. with the subsequent production of acid. Determine the various reactions for these media: MRVP broth. SIM. Citrate + and H2S Page 131 These 4 IMViC tests (actually 6 tests if you include motility and H2S) constitute. tested for by the pH indicator methyl red. and here is an additional way to determine it (although not as good as TTC). This chemical is identified when it reacts with Kovac's reagent. certainly higher priority than sugar results since they are more stable reactions. The VP is really important for identification of many bacteria. and MUST be done exactly right. The MRVP tests are run together in the same broth and then split into 2 tubes when ready to be tested for the end products. You have already tested for motility via the hanging drop slide and TTC. Simmon citrate. The amino acid tryptophan can be converted by the enzyme tryptophanase into an end product called indole.IMViC TESTS: Indole. OBJECTIVES: Understand the importance of these 4 IMViC tests. Voges-Proskauer. but for a different end product---not acid but a neutral product called acetoin (or acetylmethylcarbinol). indole production. The VogesProskauer test also determines glucose use. and motility. MATERIALS NEEDED: 1 SIM deep per unknown 1 MRVP broth per unknown 1 Simmon citrate agar slants per unknown AFTER INCUBATION: reagents • • • Kovac's reagent Barritt's reagents A (alpha-naphthol) and B (KOH) methyl red reagent . The test results from these 6 tests should carry more weight than almost any other tests. the most critical tests used for identification of bacteria after the gram stain. The methyl red test determines the use of glucose. but it is a picky test. Methyl red. since there are no other nutrients in this medium. SIM deep is a multi-test medium comprising 3 tests: sulfide (H2S gas).

Inoculate into the MRVP broth. INTERPRETATION: SIM: Indole test. you can see the red-pink color of acid presence from glucose use. • • • for methyl red: Add 6-8 drops of methyl red reagent. Voges-Proskauer tests The reagents MUST be added in the correct order. you may not even be able to see a stab line at all: it may be turbid throughout the medium. H2S. Incubate at 30 or 37 degrees C. AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: run the MR test in the tube with 2/3. and motility 1. Let sit. H2S. AFTER INCUBATION: After reading both motility and H2S. 2. a black precipitate within the agar deep. 3. Incubate at 30 or 37 degrees C. 2. 3. In fact. Record the results of your bacterial unknown in your journal. Record the results of your bacterial unknown in your journal. mix. mix. you might want to compare the inoculated SIM with an uninoculated one. the original stab line will diffuse out into the medium as the bacteria spread throughout. all the way to the bottom. 4 drops of Barritt's B. Inoculate into a tube of SIM media with a NEEDLE. 3. Citrate test 1. for Voges-Proskauer: Add 12 drops of Barritt's A. and the tube must sit undisturbed. 2. Hold the tube up to the light and look at the stab line to determine motility. 4. Lastly. 4. Methyl red Within just a few seconds after adding methyl red reagent. in the correct amounts. If MOTILE. Within just a few seconds you can see the hot pink color of indole presence. 2. FOR THAT REASON.Page 132 THE PROCEDURES: Indole test. undisturbed. Record the results of your bacterial unknown in your journal. Incubate at 30 or 37 degrees C. Methyl red and Voges-Proskauer tests 1. Streak up the slant with the inoculum. you will see the intact straight stab line. If NONMOTILE. Look for H2S. add 6-8 drops of Kovac's reagent. 3. you will add the Kovac's reagent to detect the presence of indole produced. for at least 15 minutes. and the VP test in the open tube with 1/3. and open to the air (no cap) for at least 30 minutes (45 minutes is even better) as the light pink color intensifies at the top of the tube (the . and motility 1.

BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS OF ALL OF THESE TESTS BY VISITING OTHER TABLES IN LAB.Page 133 reagents react with acetoin). . DO NOT shake the tube AFTER sitting it down for the waiting period. Citrate test The alkaline by-products of citrate use will cause the pH indicator to turn royal blue.

Why determine motility and H2S before adding Kovac's reagent? 4. 3. What are the reagents used in the indole test? methyl red test? Voges-Proskauer test? 2. The purpose of the citrate in the Simmon citrate medium is to determine if the organism can used citrate as the sole ___________ source. What is so picky about the Voges-Proskaeur test? 5.Page 134 LABORATORY REPORT SHEET QUESTIONS: 1. . The end product identified in the VP test is a neutral compound called ______.

. INTERPRETATION: Hold the plate up to the light to see the zones well. Inoculate the organisms on the plate either a straight line or a zig-zag. 2. methylene blue.LIPID HYDROLYSIS Page 135 The enzyme lipase is excreted out of the cells (an exoenzyme) into the surrounding media. Record positive reactions as weak + or strong +. There is already an indicator. 3. 4. into fatty acids which can then be taken up by the organism. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. OBJECTIVES: Identify the presence of the enzyme lipase. Run this test using your unknown bacterium. incorporated into the agar. This reaction causes the agar. a vegetable oil. normally a light blue opaque color to clear around the growth area. Incubate at 25 or 37 degrees C. A zone of clearing around the growth area identifies the presence of the enzyme lipase. Record the results of your bacterial unknown in your journal. catalyzing the breakdown of tributyrin. MATERIALS NEEDED: 1 tributyrin agar plate with methylene blue/student THE PROCEDURE: 1.

What is the substrate in this medium? 2.Page 136 LABORATORY REPORT SHEET QUESTIONS: 1. What kind of molecule is tributyrin? . 3. Name the indicator in this medium.

4.LITMUS MILK USE Page 137 Litmus is a multi-purposed and sometimes confusing medium because there are many reactions that you can get with it. OBJECTIVES: Identify the reactions associated with use of sugar and protein in milk. sometimes to the extent of coagulating the milk protein and causing curdling. the same one that is used with pink litmus and blue litmus papers in a chemistry lab to test whether a solution is acid or base. MATERIALS NEEDED: 1 litmus milk per unknown PROCEDURE: 1. Inoculate the tube of litmus milk with a loopful your unknown bacterium. or neither of these. almost looking like plasma. The major + reactions: basic reaction . The total breakdown of protein will cause peptonization. Record the results of your bacterial unknown in your journal. It starts out as a light lavender color. The use of sugar will produce acid as an end product. INTERPRETATION: BE SURE to compare your reaction against an uninoculated control from the refrigerator. also called a curd. . 3. The instructor will inoculate a set of controls showing other reactions in the litmus milk--Bacillus. Alkaligenes.pink peptonization (proteolysis) – clearing of medium acid clot/curd . The indicator is litmus. The use of the protein casein produces alkaline end products. Strep lactis.solidified whitish material BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. Incubate for up to 1 week. NOTE: It is a good idea to incubate this medium for a week before making a final decision on the reaction. both of these. The 2 nutrients in milk are casein protein and lactose sugar. 2.blue acidic reaction . hence a variety of end results. and a clearing of the medium. A bacterium can use one of these. at 30 or 37 degrees C.

Page 138 LABORATORY REPORT SHEET QUESTIONS: 1. 4. 2. You are testing the bacterium's ability to use ________ and ________ in this medium. Would a curd be an acidic or an alkaline reaction? . The production of acid means that the bacterium is using the _____ in this medium. What does a basic reaction look like on this medium? 3. producing acid as a by-product.

and N2 gas can be the end products of denitrifcation. within the Durham tube. . 25 or 37C. o A bit of zinc is about the amount that sticks to the end of a wood stick. If there is no nitrogen gas. o If you have not seen either nitrite or N2 gas. or no reduction of nitrate at all. Inoculate the nitrate broth with your bacterial unknown. Incubate at the optimal temperature. Record the results of your bacterial unknown in your journal. called denitrification. there are still a couple of possible interpretations---nitrate reduction to nitrite (NO2). Nitrites (NO2). In the absence of oxygen. various nitrogen compounds may be hydrogen acceptors: depending on which N compound is the acceptor. 2. The first obvious product of reduction to look for is reduction to N2 gas. INTERPRETATION: There are various ways that a bacterium can utilize nitrate as the final electron acceptor in anaerobic respiration. reduction to ammonia. AFTER INCUBATION: Look for N2 gas first before adding reagents. you need to add a bit of powdered zinc. the end product varies (see table below). o Add 6-8 drops of nitrite reagent A. OBJECTIVES: Identify the different ways that nitrate can be reduced by bacteria. o Add the same number of drops of nitrite reagent B. for your organisms. 4. ammonia (NH3). MATERIALS NEEDED: 1 nitrate broth per unknown AFTER INCUBATION: nitrate reagents A (sulfanilic acid) and B (naphthylamine) wooden sticks for zinc zinc powder THE PROCEDURE: 1. 3.NITRATE TEST Page 139 Nitrate (NO3) reduction can be performed by many bacteria. o You should see a reaction within a minute or less.

Name the 2 major end products of nitrate reduction. LABORATORY REPORT SHEET QUESTIONS: 1. How do the definitions of nitrate reduction and denitrification differ? 4.no reaction none yes none none red no color no color no color -. WHY add zinc powder? WHEN do you add it? 3.Page 140 REACTION N2 gas Color after adding reagents Color after adding zinc NO3 to NO2 NO3 to N2 NO3 to ammonia NO3 .(not added) no color no color pink-red BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. 2. Is nitrate reduction an aerobic pathway or an anaerobic pathway? .

• Pick your inoculum. not with a metal loop (reagent may react with the metal). ask for a new tube from the instructor • Use a young culture. . and will be a bluish-purple color that progressively becomes more purple. it is old and should not be used). The oxygen will change the reagent color as time passes. • Read the reaction within 20 seconds (NOT after). A couple of key points when doing this test: • We keep the oxidase reagent either frozen or unopened in tubes until needed. • Use a culture growing on an agar plate or agar slant. • Use FRESH reagent. preferably less than 24 hrs old. DO NOT READ the reaction after 20 seconds. MATERIALS NEEDED: oxidase reagent (Tetramethyl-p-phenylenediamine) wooden rods THE PROCEDURE: 1. 2. 3. Pick a good-sized amount of inoculum from a plate culture or slant culture and place it on a piece of filter paper. usually it will change in less than 15 seconds. Add one drop of the reagent (if it is dark blue. The enzyme cytochrome oxidase is involved with the reduction of oxygen at the end of the electron transport chain.OXIDASE TEST Page 141 The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -). reacting with oxygen. so it must be read quickly. If old reagent is sitting out on the bench and is PURPLE. turn a color. but with a wooden stick. The colorless reagent will detect the presence of the enzyme oxidase and. INTERPRETATION: A positive reaction will usually occur within 10-15 seconds. OBJECTIVES: Test for the enzyme oxidase on your unknown isolates. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS. TIME the reaction: a positive reaction will occur within 20 seconds. less than a couple of hours old (it is taken out of the freezer).

Page 142 LABORATORY REPORT SHEET QUESTIONS: 1. Why do you have to read this reaction within 30 seconds? 2. Why does the oxidase reagent need to be fresh? .

there will be less and less starch to react with the iodine. .STARCH HYDROLYSIS Page 143 The enzyme amylase is secreted out of the cells (an exoenzyme) into the surrounding media. Incubate at either 25 or 37 degrees C. MATERIALS NEEDED: Gram's iodine reagent (AFTER incubation) 1 starch agar plate THE PROCEDURE: 1. the starch will not have been degraded: the medium will be purple. catalyzing the breakdown of starch into smaller sugars which can then be absorbed by the cells for use. INTERPRETATION: Placing the agar plate on a white piece of paper or background will REALLY help you to distinguish the zones. Make a single line streak of the unknown bacterium across the plate. Strong amylase producers may convert all of the starch in the agar to sugars. 2. producing a deep purple color. 3. In the absence of amylase. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB. As starch is catabolized and converted to sugars. AFTER INCUBATION & GROWTH: Flood the plate with iodine Record the results of your bacterial unknown in your journal. Iodine reacts with starch. there will be a yellow/gold zone AROUND the growth. In the presence of the enzyme amylase and subsequent starch hydrolysis around the growth area. OBJECTIVES: Identify the reactions associated with growth on a starch agar plate. while weak amylase producers may convert the starch surrounding the growth areas only. 4.

WHY? 2.Page 144 LABORATORY REPORT SHEET QUESTIONS: 1. 3. Starch hydrolysis will result in a zone around the bacterial growth that is the color _____. What kind of molecule is starch? . The enzyme that does this is called ________.

MATERIALS NEEDED: 1 urea broth/per unknown THE PROCEDURE: 1. 25 or 37 degrees C. 3. Inoculate a tube of urea broth with your unknown bacterium. INTERPRETATION: The alkaline reaction turns the pH indicator to hot pink. producing the alkaline product of ammonia. A yellowish color is still a negative reaction. . 2. although acidic. Record the results of your bacterial unknown in your journal. That causes the phenol red pH indicator phenol red to turn a beautiful shade of hot pink. BE SURE THAT YOU SEE BOTH – AND + TEST RESULTS BY VISITING OTHER TABLES IN LAB.UREA HYDROLYSIS Page 145 Urea (a protein compound) can be broken down with the help of the enzyme urease. Incubate until next period at optimal temperature. OBJECTIVES: Understand the reactions of bacteria in urea broth.

3. 2. Name the indicator in this medium. OR neutral chemical? Name it. Is the end product being tested for an acidic. basic. What type of organic compound is urea? .Page 146 LABORATORY REPORT SHEET QUESTIONS: 1.

Inoculate a large colony (2-3mm diameter)of the bacterium (pure culture) into the 0.API 20E Page 147 This API-20E test strip (from bioMerieux. Barrett's reagents A and B. Inc.) is used to identify the enteric gram negative rods (although API makes a variety of other test strips for yeast. A profile number is determined from the sequence of + and . Some of the wells will have color changes due to pH differences: others produce end products that have to be identified with reagents. E. then looked up in a codebook having a correlation between numbers and bacterial species.g.85% NaCl solutions (5ml) sterile Pasteur pipettes + bulbs sterile mineral oil API 20E test strip (for oxidase . 2. This particular strip is for enteric (intestinal) bacteria only.test results. making sure that the suspension is homogenous and without clumps of floating nbacteria. Use a McFarland barium sulfate standard #3 to quantitate the suspension. all dehydrated. e. A bacterial suspension is used to rehydrate each of the wells.gram negative rods) API test strip incubation chamber AFTER INCUBATION: 10% FeCl3. MATERIALS NEEDED: agar plates of bacterial species 0.) 20 separate test compartments are on the strip. Kovac's reagent PROCEDURE: Prepare a suspension of the bacteria in the saline tube 1. etc. OBJECTIVE: Learn how to perform and interpret the miniaturized. anaerobes. coli. . Staph.85% NaCl solution. multi-test technique for bacterial identification.

and GEL have boxes around their names. 3.htm A more specific 9-digit number can be obtained with a few more tests:. Place the top of the incubation chamber over the bottom. and is added as the last test result.com/industry/food/api/apiweb. 4. 4. you will now inoculate the bacterial suspension into each well with the sterile pipette. Three test reactions are added together at a time to give a 7-digit number. The bottom of the incubation chamber has small indented wells in the bottom: fill it with water just enough to fill these indentations. Holding the strip at a slight angle up from the table top. . These test wells will be filled all the way up to the top of the well. CIT. Each well should be filled up to the neck (see diagram). Touch the end of the pipette to the side of the cupule. 2. Incubate the strip in its chamber 1. Place the strip into this bottom.biomerieux-usa. but they will then be filled up to the top with sterile mineral oil. 2. allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. Record results on the diagram handed out to you in lab (1. 0 points for . and URE are filled as described in step B. or 4 points for + reaction. 2. 3. LDC. 4. H2S. This should eliminate any bubbles forming in the wells. but NOT DURING THIS LAB. Place the strip at 37o C for 18-24 hours. and label it. ODC. The oxidase test reaction should be negative.reaction). There should not be so much water that it slops onto the API strip. Add the proper reagents to the compartments: o 1 drop of Kovac's to the IND (read within a couple of minutes) o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes) o 1 drop of FeCl3 to TDA 2. which can then be looked up in the ONLINE codebook at http://industry. 3. ADH.Page 148 Inoculate the API strip 1. INTERPRETATION: 1. Read all other tests as described (chart below) without reagents. VP.

) pink/red (10 min.) black diffuse yellow yellow yellow yellow yellow yellow yellow yellow yellow violet . from BioMerieux) READING THE API 20 TESTS ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA OX SUBSTRATE ONPG arginine lysine ornithine citrate Na thiosulfate urea tryptophan tryptophan Na pyruvate charcoal gelatin glucose mannitol inositol sorbitol rhamnose sucrose melibiose amygdalin arabinose oxidase REACTION TESTED beta-galactosidase arginine dihydrolase lysine decarboxylase ornithine decarboxylase citrate utilization H2S production urea hydrolysis deaminase indole production acetoin production gelatinase fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation fermentation/oxidation oxidase .RESULTS colorless yellow yellow yellow pale green/yellow colorless/gray yellow yellow yellow colorless no diffusion of black blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green blue/blue-green colorless/yellow + RESULTS yellow red/orange red/orange red/orange blue-green/blue black deposit red/orange brown-red red (2 min.Page 149 (pictures from the API 20E documentation.

What are the advantages of this test (compared to regular biochemical tube media)? 4. What are the disadvantages of this test? 5. Did your table’s unknown bacterium correctly identify? .Page 150 LABORATORY REPORT SHEET QUESTIONS: 1. What is the function of the mineral oil? 3. What is the purpose of the water in the tray? 2.

prosthetic joint infections. Staphylococcus is not halophilic. epidermidis and S. A selective medium has an inhibitory agent which favors the growth of certain bacteria by inhibiting others. It can produce a range of toxins including enterotoxins (food poisoning). OBJECTIVES: Obtain presumptive staphylococci from nasal specimens Isolate and identify different staphylococci species using selective and differential agar MATERIALS NEEDED: Sterile swabs Sterile saline solution 2 Mannitol salt agar (MSA) plates (only 1 used for 1st session) Containers of alcohol + forceps . coagulase. hominis but the first three are the most common isolates. The high salt content in SM110 and MSA inhibits other common skin microorganisms. When stained. saprophiticus) are much less frequently found as pathogens but are occasionally associated with endocarditis. This exercise gives you the opportunity to use selective media. aureus is able to cause many superficial pyogenic (pusforming) infections of the dermis and underlying tissues as well as serious systemic infections. aureus is often considered to be the most problematic of the three pathogens and is distinguished from the other two by being the only one able to coagulate plasma. S.) belonging to the family Micrococcaceae that are often found as normal human microbiota of the skin and nasal cavity.5% NaCl). including toxins.8 – 1. which makes it a differential media also. In a previous exercise. Staphylococcus is usually either beta hemolytic or not hemolytic at all (called gamma hemolysis). S. you learned about halophilic bacteria. Not only salt resistant. and S. S. saprophiticus S. but rather haloduric (salt tolerant). in that it can live in or endure high NaCl concentrations (but does not require the salt). MSA contains an additional indicator for monitoring mannitol fermentation. Pathogenic Staphylococci can produce a variety of virulence factors. S. epidermidis. and for identification of the species.0 µm dia.GENUS STAPHYLOCOCCUS: Isolation and Identification Page 151 Staphylococcus is a genus of Gram +. non-spore-forming cocci (0. haemolyticus. The other media being used in this exercise are for differentiating pathogenic Staphylococcus from nonpathogenic.7. Staphylococcus is always facultatively anaerobic. and hydrolytic enzymes that can damage host tissues. There are five organisms to consider as potential human pathogens in this genus: S. aureus. it will be seen in small clusters (staphylo = cluster). wound infections and infections of cerebrospinal fluid. in this case based on high sodium chloride (MSA and SM110 are both selective media for the isolation of Staphylococci. and toxic shock superantigens. cytotoxins (general systemic toxins). The other coagulasenegative staphylococci (S. leucocidins.

2. Aseptically remove a cotton tipped swab from its wrapper and dip into a tube of sterile saline. Divide a Columbia naladixic acid (CNA) agar plate and another MSA plate in half. Remove excess saline by pressing against the tube wall and rotating the swab. 4. 1st Session 1. they will then be sterile. Place the forceps into the alcohol and then sterilize the forceps by running them through the flame quickly. Identify 2 colonies from the 2 MSA plates that fit this Staph profile. Another characteristic you should look for is any colony around which the medium has turned yellow (identifying the fermenters of mannitol. You may now pick a novobiocin disc from the holder to place on the opposite sides of the CNA plate. 2. 2nd Session 1. You will use a dense zig-zag inoculation (see above diagram) to streak the 2 potential Staph colonies on opposite sides of the CNA and MSA plates. of which some species of Staph are). each specimen streaked onto a mannitol salt agar plate. . 4. Use the moistened swab to collect bacteria from the anterior opening of the nostrils. Incubate plates at 37°C or room temperature (if over the weekend). Your aim is to isolate a Staph from your MSA plate.Columbia naladixic acid blood agar plates (CNA) Staphylococcus medium 110 (SM110) agar plates DNase agar plates Novobiocin (5 microgram) antibiotic disks Metric rulers Rabbit plasma (frozen) for coagulase test Staphylococcus identification tables Optional media for identification: arginine and ornithine decarboxylase broths urea broth VP broth SCHEMATIC OF IDENTIFICATION PROCEDURE 2nd period 3rd period 1st period MSA from CNA and MSA gram stain Nasal catalase SM110 DNAse Page 152 4th period 5th period coagulase finish test reading optional tests prepare report TSA (Staphyloslide) THE PROCEDURES: Be sure to keep a list of all test results for your isolates. 3. 3. Be SURE that you have only 1 colony on the loop for this transfer. although not the only genus of bacterium that will grow in high salt. The alcohol will catch on fire and when evaporated from the forceps. using salt resistance as a key indicator. Staphylococcus is salt resistant. Using a DENSE zig-zag inoculation. Two people on the table will perform nasal swabs. inoculate a MSA plate and incubate at 37°C or room temperature (if over the weekend).

4. Since there are 2 kinds of coagulase enzyme—bound and free---there are 2 different tests that can be used to identify these enzymes. 3. called a partial hemolysis • beta (β) hemolysis – complete clearing around colony caused by breakdown of RBCs by streptolysin enzymes • gamma (γ) hemolysis .. in different ways. You will carry through the rest of this exercise with your table’s 2 Staph isolates. Nutrients and vitamins in this medium enhance the pigmentation of the pathogenic Staphylococcus. Presumptive S. Gram stain the 2 colonies (taken from CNA plate) to verify that you have gram + cocci in clusters. you are checking for 2 reactions---sensitivity to novobiocin antibiotic AND hemolysis of blood. Staph aureus produces alpha toxin which typically causes wide zones of beta (complete) hemolysis. Check DNase plates for hydrolysis. You will need to do a gram stain and catalase test as if it were your own isolation from your nasal cavity. • If DNAse. the most reliable. On the CNA plate. 2. or you can “borrow” another table’s isolate. 5. Novobiocin sensitivity . meaning that they are salt resistant. If growth is present. . no zone Staphylococcus species are either beta hemolytic or gamma (not hemolytic). If your isolate is not this profile. What you do with your Staph isolate is now determined by its reactions on SM110 and DNAse. If your isolate is not this profile. a. Check the SM110 for growth and for pigment. those colonies becoming a yellow-orange colony.and SM110 –.Page 153 3rd Session 1. Both of the enzymes activate fibrinogen in plasma. 6. Observe the MSA plates noting IF colonies grow. b. a. that will be the end of the isolate’s use in this exercise. that will be the end of the isolate’s use in this exercise. 2. 3. IF NEITHER OF YOUR TABLE’S ISOLATES ARE STAPHYLOCOCCUS. aureus can be confirmed using the coagulase test. 4th Session 1. If the medium surrounding the colonies has changed from red to yellow. mannitol has been fermented and the phenol red pH indicator in the medium has changed colors as a result of the acid from sugar breakdown. Using your Staphylococcus isolates.A zone 16 mm or larger indicates sensitivity b. Hemolytic reactions • alpha (α) hemolysis – green zone around colony. ask your instructor to give you an unknown Staph to work with for the rest of this exercise. run the coagulase test on the isolate 4. streak 2 agar plates---SM110 and DNAse (divide each in half). Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase + (all species of Staph are +). then the second question is whether the bacterium uses the sugar mannitol. Incubate plates at 37°C or room temperature (if over the weekend). caused by leaking hemoglobin converted to biliverdin. The TUBE method is the definitive test of the 2. • If DNAse + and SM110 +.no hemolysins. Refer back to that DNAse exercise.

To interpret. There will a visible clumping of cells within 10-15 seconds. Any degree of coagulation is considered a positive test for the free coagulase enzyme. 6. Inoculate the following media. digital camera) .g.Page 154 • THE SLIDE AGGLUTINATION TEST Make a 1 inch diameter circle on a clean glass slide using a wax pencil. include: • All test results • A flow chart/dichotomous key for identification • A diary/journal of this activity (include visuals of the test results. read your reactions the NEXT lab period. e. 5. assuming that your species are common ones. This test is for the bound coagulase enzyme. • Urea broth • Voges-Proskauer broth (see MRVP in IMViC exercise) • Ornithine decarboxylase (see Decarboxylation and Deamination exercise) • Arginine decarboxylase (see Decarboxylation and Deamination exercise) Differentiation of S. looking for solidification of the plasma. aureus S. or clump. For both isolates. There are enough tests that have been performed to identify your Staphylococcus isolates. Incubate at 37°C or room temperature. The following table has those 3 species listed with the corresponding test results. aureus from other common staphylococci microbiota S. Place at 37C and check at the next lab period. Your table will write up this exercise—1 report for the table. and refer to the Staph identification tables available in lab. 7. • THE TUBE COAGULATION TEST Inoculate a tube with a ½ ml of rabbit plasma with the bacterial inoculum. saprophiticus + (most strains) Alpha toxin (β-hemolysis) Acid from mannitol + + (most strains) Coagulase reaction + Pigment production (SM110) Usually golden Usually white Usually white DNase production + Sensitivity to novobiocin sensitive sensitive resistant 8. Place two drops of thawed rabbit plasma into the circle and using a wooden pick or a clean loop. you can run the following media (we have used all of those in other exercises). epidermidis S. If you have Staph aureus. causing them to agglutinate. If your isolate does not match the profile of the 3 species of Staphylococcus shown in this table. tip the tube at an angle. Fibrin threads form between the cells. Try to identify your isolates. Add a single colony and emulsify it in the plasma. Word or another word processing program is the best procedure because you can embed pictures into the document. subculture the bacterium onto a TSA plate to run the Staphyloslide test coming up in lab.

newborn infections.GENUS STREPTOCOCCUS: Isolation and Identification Page 155 Streptococci are part of normal human microbiota. toxic shock. Streptococcus is facultatively anaerobic or aerotolerant. As with the Staphylococci. so it is important that you understand the categories of hemolysis. but generally favoring high carbon dioxide levels (hence. The most significant pathogens are Streptococcus pyogenes (Group A). impetigo. you will be using a selectively differential medium called mitis-salivarius agar. as well as exotoxins. Streptococcus agalactiae (Group B) and Streptococcus pneumoniae. found on the skin. in the upper respiratory tract. oralis---associated with dental plaque • Mutans—Strep mutans. it will be seen in pairs or chains that vary from a few to dozens. Streptococci are Gram-positive cocci which grow primarily in pairs and in chains of cells. and necrotizing fasciitis. . When stained. gordonii. and streptolysins. the use of the candle jar for incubation). Streptococci have a wide variety of virulence factors---enzymes such as hyaluronidase (spreading factor for necrotizing fasciitis). and it is one of the organisms that we will try to identify. • alpha (α) hemolysis – green zone around colony. digestive tract and oral cavity. intermedius—in gingival crevices The most common species of Streptococci in the oral cavity are the ‘viridans’ group---the alpha hemolytic species. Tellurite and crystal violet dye inhibit gram – bacteria as well as most gram +. M-S agar has sugars. vestibularis—covering mucosal surfaces • Anginosus/milleri—Strep anginosus. Because there are so many species of different bacteria genera in the mouth (more than 200 species have been cultured and identified). Streptococcus mutans is the leading cause of dental caries in humans. Hemolysis is a very important feature used to identify the streptococci. The group D Streptococci are resistant to extreme temperatures. and the dye trypan blue produce different color. and are a hardier group than most of the other Streps. The pygenic Streptococci group contain the pathogenic species. Some streptococci (formerly group D) now placed in the genus Enterococcus are of medical significance because they are often difficult to treat because of antibiotic resistance. The genus is always catalase negative. particularly sucrose. that give a luxuriant growth of the Strep species. These species will be beta hemolytic. sanguis. sobrinus—associated with plaque and dental caries • Salivarius—Strep salivarius. leucocidin. Streptococci are often grouped by hemolytic reaction on blood agar and by serological typing using the Lancefield classification system. caused by leaking hemoglobin converted to biliverdin • beta (β) hemolysis – complete clearing around colony caused by breakdown of RBCs by streptolysin enzymes • gamma (γ) hemolysis The common oral Streps fall into 4 categories: • Mitis—Strep mitis. ones causing strep throat.

Two specimen types will be taken---a throat specimen and a specimen from the toothgum area. SXT. cause a wide range of superficial skin infections along with more serious diseases like pneumonia.5% NaCl 4th period 10 and 45 C incubation THE PROCEDURES: Be sure to keep a list of all test results for your isolates. b. OBJECTIVES: Obtain presumptive streptococci from throat swabs and attempt to isolate from blood agar plates Using isolates to identify different types of hemolysis Perform testing to narrow down the identification of the streptococcal isolate MATERIALS NEEDED: Sterile cotton sawbs Tongue depressers CNA Blood agar plates containing naladixic acid Containers of alcohol + forceps Blood agar plate (5% sheep red blood cells) mitis-salivarius agar plate (M-S) Sensitivity disks (Bacitracin. meningitis. like staphylococci. 2..5% NaCl broth TSB SCHEMATIC OF IDENTIFICATION PROCEDURE 2nd period 3rd period 1st period Throat culture . Optochin) 3 Bile esculin slants 3-6. Streptococci. tongue or teeth. using 3 or 4 sections). with the exception of using a cotton swab for the first section and then switching over to the . The procedure is identical to a streak plate procedure. Use a tongue depressor to push the tongue down so a sterile swab can be used to obtain a sample from the back of the throat (pharynx). erysipelas and necrotizing fasciitis. Avoid swabbing the cheeks. The subject should tilt the head back. Cover one third of a Columbia naladixic acid blood agar (CNA) plate with the throat swab Then use a sterile loop to make a standard ISOLATION STREAK PLATE. a. 1st Session 1.blood agar blood agar gram stain Tooth scraping – M-S agar catalase Bile esculin 6. Run the cotton swab along the gum-tooth line. or gamma hemolytic. Try to get back back in the area of the molars or molar/premolars to get the tooth culture.Page 156 Streptococcus can be beta. alpha.

5% NaCl. The reduced oxygen atmosphere will encourage good hemolytic reactions and provide more luxuriant growth of Streptococcus. IF YOU HAVEA BETA HEMOLYTIC COLONY. granular “frosted glass” • Strep sanguis smooth. 3. place a bacitracin disc on the first section of the plate (using alcohol-flamed forceps). Using the swab from the tooth-gum area.no hemolysins. On each side of the BAP.Page 157 inoculating loop. that will be the end of the isolate’s use in this exercise. Strep pneumonia is sensitive to optochin. The BAP will be divided in half—with each of your 2 table’s isolates streaked in a dense zig-zag pattern. 5. and it is normal flora in many people. • Strep mitis tiny blue • Strep salivarius large. no zone 2. • Identify the type of hemolysis (alpha or gamma) • Determine the sensitivity of the isolates to the 2 discs o ANY zone around the SXT disc means sensitivity o A zone around the optochin disc must be at least 14mm. 5. It is imperative that you get good isolation of colonies for this exercise. Do not get the 2 isolates too close to each other on the plate. .5% sodium chloride broth. 3rd Session FOR COMMON ORAL STREPTOCOCCI 1. If your isolate is not this profile. Space the 2 discs out enough so they are not too close together. In addition. Use the same technique described above in step 2. Inoculate this bacterium into those 2 media along with your own isolates. • alpha (α) hemolysis – green zone around colony. 4. 3. Check the throat culture for hemolytic zones. Check the growth of your Strep isolates on the blood agar plate. 2. hard colonies embedded in agar 4. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase (all species of Strep are -). aseptically place an optochin disc and an SXT disc (using alcohol-flamed forceps). on top of each streaked isolate. streak a mitis-salivarius agar plate. Gram stain the 2 colonies (taken from BAP) to verify that you have gram + cocci in pairs or chains. partial hemolysis • beta (β) hemolysis – complete clearing around colony • gamma (γ) hemolysis . 3. mucoid colonies • Enterococcus fecalis blue/black • Strep mutans raised pale blue. 4. If your isolate is not this profile. that will be the end of the isolate’s use in this exercise. Identify 2 different colony types on the M-S agar to streak to a blood agar plate. light blue “gumdrop”. It is not likely that you will find a beta hemolytic since those species are pathogenic. Use the BAP to streak the surface of a bile esculin slant and to inoculate a tube of 6. 2nd Session 1. Incubate the plates in a candle jar at 37°C. IN ADDITION. Check the mitis-salivarius agar plate for different kinds of colonies. subculture the colony onto a new blood agar plate using proper islation streak technique. each table will be given a group D Strep as a + control for bile esculin and 6.

Check the sensitivity of the isolate to bacitracin. 5. Incubate the test til the next period. pneumoniae Group A B D D viridans Hemolysis beta beta alpha or gamma alpha. Enterococcus will grow at these temperatures. This is a + test. agalactiae E. Organism S. and inoculate. These are both + test results. Bile esculin interpretation: Growth on the medium means that the bacterium is resistant to 40% bile. salivarius S. 5. sobrinus can be γ alpha Bacitracin SXT S R R R R R R R S S Optochin Bile: esculin R R R R R 6. as well as resistant to high salt.=no growth We have esculin sugar (without bile) available as a test. those species that use the sugar esculin will turn the medium dark brown/black (the color of black coffee). Incubate the tests at 37°C. 3. mutans S. . gamma Usually α -Salivarius. The group A Strep are sensitive to bacitracin. Be sure to place the plate into a candle jar. Look for a dark brown/black color change. 2. b. mutans. that will be the end of the isolate’s use in this exercise. a. If the bacterium is resistant to high salt. bovis S. Use the chart below to presumptively identify your streptococci.6. Add ½ ml of BHI broth to a sterile test tube. If your isolate is not this profile. Read any tests that were run last lab session.5% sodium chloride broth. 4. 2. Incubate the tests at 37°C. it will grow and the NaCl medium will be cloudy. you will use this same culture for the rapid Strep test that is upcoming in lab. pyogenes S. you will inoculate 2 tubes of TSB and place the 2 tubes at 10C and 45C until the next lab period. Page 158 IF YOU HAVE A BETA-HEMOLYTIC STREPTOCOCCUS 1. Any zone of inhibition shows sensitivity. 4th Session 1. aseptically add the esculin disc. Run a catalase test (see Catalase test exercise) to verify that your isolate is catalase (all species of Strep are -). So if your slant is dark brown/black. mitis S. it means that it uses esculin as well as being resistant to bile. you likely have a group D Enterococcus. Refrigerate the culture until that lab. that will be the end of the isolate’s use in this exercise.faecalis/faecium S. Read the NaCl and bile esculin tests.5% NaCl 10/45C growth + + - : : : + : + : - + - + (can be + if testing esculin alone) -R/S S S - : - - R =resistant S =sensitive + =growth . ask your instructor to get you a disc. If your isolate is not this profile. If you had a beta hemolytic Strep that fits the Strep profile (correct gram stain and catalase result). Gram stain the culture to verify that you have gram + cocci in pairs or chains. Use the BAP to streak the surface of a bile esculin slant and to inoculate a tube of 6. If you want to run esculin sugar alone. In this case. 3. In addition. If your isolate is + for bile resistance and esculin use. 4.

using the terms “viridans” and “color. Name two autoimmune diseases associated with the group A beta streptococci. Why is the optochin test important for use on alpha streptococci? 4. Why is a bacitracin (A) disk so important in strepotococcal identification? What are the consequences of not making a diagnosis of bacitracin sensitivity? 2. What does a hemolysin do? .” There is a group of Strep called the Viridans group. 3. What category of hemolysis? 5. Do a search using Google on the web.Page 159 LABORATORY REPORT SHEET QUESTIONS: 1.

Page 160 .

producing a noticeable agglutination (which is more easily seen with the blue of the latex). This is a good confirmation of S. and a POSITIVE control are each placed on the glass slide. OBJECTIVES: 1. Blue latex powder pre-coated with the antibody to Staph aureus is mixed with a Staph culture. This exercise involves 2 kinds of serological tests---one for the identification of an unknown antigen and the other for the identification of an unknown antibody. To make a serological identification using Staphylococcus previously isolated and identified by biochemical testing. 2. Another way to use serological testing is for the identification of the antigen itself. It differentiates Staphylococci which possess clumping factor and/or Protein A. If antibodies are present in the serum or plasma and mixed with the latex. The latex particles in the MONO-LATEX REAGENT LATEX are coated with a suspension of mononucleosis antigen obtained from red blood cells from cattle. Any feature that can elicit an immune response is called an antigen. Burkitt's lymphoma. which are very different from the ones inducing the antibody formation. The patient serum. called ‘heterophile' antibody reacts against sheep and bovine RBCs. The term heterophile antibody refers to antibodies having the capacity to react with certain antigens. If the specific antigen of Staph aureus is present. This odd antibody. MATERIALS NEEDED: Staphylococcus strain isolated in the Staphylococcus experiment . and nasopharyngeal cancer). a NEGATIVE control. for the identification of patient antibody to this virus (which causes infectious mononucleosis.SEROLOGICAL TESTINGLATEX AGGLUTINATION Page 161 The surface of microorganisms is covered with physical features that can be recognized by the immune system. The interesting thing about this test is that you are identifying NONSPECIFIC antibody rather than antibody against the Epstein-Barr virus. The immune system makes proteins called immunoglobulins or antibodies which bind to an antigen to either directly neutralize the antigen or cause it to be cleared more efficiently by other components of the immune system. aureus that has been presumptively identified by biochemical and physical testing. the cause of infectious mononucleosis. it will react with the latex-antibody. then adding the reagent LATEX (plastic beads covered with antigen). Such agglutination is directly observable to the naked eye as a clumping reaction. In the Monospot test. using a known specific antibody for that antigen: a latex-conjugated antibody (Staphyloslide kit) can be used to confirm the presence of Staphylococcus aureus. a known Epstein-Barr viral antigen is used in the test. a specific reaction will result in a visible agglutination of the latex particles. To identify non-specific antibodies (heterophile antibodies) against Epstein-Barr virus. Antibodies linked to large particles such as latex beads can be used to agglutinate microorganisms using specific reactive antibodies.

Dispense 1 drop of Test Latex (contains Staph antibody) onto one of the circles on the reaction card and add 1 drop of Control Latex onto another circle. 5. Be sure you have a homogenous bacterial suspension. Serological identification of beta-hemolytic Streptococcus group A This test is based on the same antigen-antibody reaction as the Staph . 3. 4. expel any latex from the dropper for complete mixing. 7. Pick up and hand rock the card for up to 20 sec and observe for agglutination under normal lighting conditions. using the same bacterial culture. Read macroscopically 6. Repeat step 3 for the Control Latex. Rock the slide back and forth for no longer than one minute and look for blue agglutination. Clumps will make the reaction difficult to identify.Page 162 BD Staphyloslide kit Monospot test kit for Epstein-Barr virus Clearview Exact Strep II dipstick kit cultures of Staph aureus and Strep group A to use as + controls THE PROCEDURES: Serological typing of Staphylococcus aureus 1. Using a microbiological loop pick up and smear 5 suspect colonies (either a suspect Staph from your body OR a known Staph aureus supplied from the lab) onto the Test Latex-containing circle and mix this into the Test Latex reagent. Spread to cover the circle. aureus. Clumping is a positive reaction for the identification of S. Dispose of the reaction card in an appropriate biohazard container. Mix the latex reagent by shaking. 2. and possibly give a false + reaction.

If you isolated a suspect group A Strep from your throat. 4. cytomegalovirus. leukemia. Place a free-falling drop of NEGATIVE control serum on the glass slide 2. Do not touch the reagent to any of the drops already on the glass slide. Observe for agglutination. covering the entire surface of the circle. and rheumatoid arthritis. Since we don't have patient serum. . you will use the positive control serum as the patient's serum. The test must be read at 2 minutes. 5. Monospot test 1.Page 163 There will be available cultures of Strep group A so that you can see what a + result looks like. Heterophile antibodies have also been identified with Burkitt's lymphoma. 3. Add 1 free-falling drop of the reagent LATEX to each well. Rock the slide in a circular motion for 2 minutes (40 rocks per minute). Mix the contents of each circle with a stirrer (different stirrers for each circle). Place a free-falling drop of POSITIVE control serum on the glass slide in a different well. A light held directly over the slide will improve the readability of the test. you may use that culture. 6. viral hepatitis.

are you testing for a patient’s antibodies to Staph OR the antigen itself? .Page 164 LABORATORY REPORT SHEET QUESTIONS: 1. If antibodies are regarded as being antigen-specific how can you explain the occurrence of heterophile antibodies in mononucleosis? 4. Why are the antibodies attached to latex particles for this type of test? 3. In the Staphyloslide test. What feature of antibodies results in a positive test causing clumping? 2.

arginine) DNAse gelatin hydrolysis H2S indole test lipid hydrolysis methyl red test salt resistance (7.5%) mannitol use tolerates bile BIOCHEMICAL TESTS – FAQs NAME OF MEDIUM INDICATOR or REAGENT iron dark coffee slant + REACTION (visible reaction) any growth bile esculin slant uses esculin sugar Page 165 . lys.BIOLOGY 2420 PURPOSE NAME OF TEST bile resistance esculin hydrolysis casein hydrolysis catalase coagulase test citrate test deaminase (phenylalanine) decarboxylase (orn.REACTION (visible reaction) no growth no color change no specific medium methylene blue mannitol salt agar .

Page 166 litmus milk (different reactions) TTC motility agar nitrate reduction oxidase test ONPG test phenol red sugar broths starch hydrolysis sugar fermentation (various discs) urea hydrolysis voges-proskauer .

colonies CHEMICALLY-DEFINED MEDIUM a synthetic medium. non-self immunogenic material that elicits an immune response ASEPTIC TECHNIQUE procedure to guarantee sterility and to reduce contamination ATRICHOUS without flagella. containing 210% O2 and around 10% CO2 CFU colony-forming units.Page 167 GLOSSARY OF TERMS AEROBIC requires oxygen (opposite of anaerobic) AGAR powder added to media for solidification AIR-DRY drying of slide suspension in air before heat fixing and staining AGGLUTINATION the reaction between antigen and specific antibody ALIQUOT dispense an amount of liquid using a pipette ANALOG similar structure. but not identical ANTIBODY specific. comprised of known ingredients and known amounts CHITIN a complex polysaccharide molecule found in walls of fungi and exoskeletons of shrimps and crabs CHROMATOGRAPHICALLY migration and separation of molecules through an absorbant material like cotton. over time . protective protein produced by the immune system in response to an antigen ANTIGEN foreign. i. used for stained and live specimens BROTH medium without agar BROWNIAN MOVEMENT vibrations of an object seen in a microscope.e. nonmotile AUTOCLAVE moist heat method of sterilization using pressure AXIAL FILAMENT a structure for motility used by the Spirochaete bacteria BIBULOUS PAPER absorbant paper used to blot dry slides after staining BHI brain heart infusion. a really good enrichment medium BRIGHTFIELD MICROSCOPY full light directed towards the specimen. not true motility CANDLE JAR candle burns in a closed container producing a carbon dioxide incubator.

e. in feces FLAGELLA a structure for motility FLAGELLATION differential category of flagella placement around the cell GENUS category of organisms with like features and closely related. requiring grow factors or particular nutrients FECAL COLIFORMS gram . divided into species HALOPHILIC salt-tolerant or salt-loving (salt-requiring) . used for wet mounts DECOLORIZER the reagent used to remove the primary dye from the cell wall in a differential stain. nonsporeforming. e.g.g. methylene blue DARKFIELD MICROSCOPY special condenser blocks most light rays.g. e. safrinin. selective (for Gram -) and differential medium EXOENZYME enzyme excreted away from the cell FACULTATIVE ANAEROBE uses oxygen when present but can either ferment or anaerobically respire without it FASTIDIOUS hard-to-grow bacteria.Page 168 CNA Columbia naladixic acid medium. selective (for Gram +) and differential medium COAGULATION clotting of blood. GI flora in animals. acid alcohol.g. i.rods which ferment lactose. plasma COLIFORMS gram . nonsporeforming COLONY a visible mass of bacteria growing on solidifed medium.rods which ferment lactose. lactose or sucrose EMB Eosin methylene blue medium. acetone-alcohol DIFFERENTIAL STAIN uses 2 or more dyes which allow differentiation between different bacterial groups or structures DIAPHRAGM acts as an iris within the condenser which opens and closes for sensitive light control DIPLOID A full complement of chromosomes (e. blood agar CONDENSER collects all available light rays for direction up to the stage opening COUNTERSTAIN the 2nd dye added to a smear. 46 or 23 pairs for human) DISACCHARIDE 2 sugar molecule. only reflected light bounces off specimen into lens producing a black field of vision.e. taken in after the wall is decolorized. a clone COMPLEX MEDIUM medium with some unknown ingredients or amounts.

e.Page 169 HAPLOID Half the normal number of chromosomes HEAT-FIX use of the flame to 1)coagulate proteins of the suspension. skeleton of the cell MONOSACCHARIDE simple sugar. Voges-Proskauer. lenses alignment PATHOGENIC disease-causing PCA plate count agar medium.g. and 2) kill the microbes HELMINTH parasitic worm HYPHA A fungal filament (plural. e. to be used always with immersion oil PALLINDROME a word or a group of letter that is read frontwards and backwards the same. radar PARFOCAL feature of microscope which allows rotation of lenses with only minor focus movement. general all-purpose enrichment PFU plaque-forming units produced by bacterial viruses when infecting host bacterial cells PHASE-CONTRAST MICROSCOPY special condenser below stage and in objectives change speed of light rays. glucose MYCELIUM A visible mass of hyphal filaments NA/NB nutrient agar or nutrient broth NONIONIC no electrical charge OBLIGATE AEROBE requires oxygen to grow OBLIGATE ANAEROBE does not use oxygen to grow. framework. enhancing density differences inside and outside of cells. may even be killed by it OIL-IMMERSION LENS 100X objective lens.g. citrate INOCULUM a small sample of the microorganism MIC minimal inhibitory concentration of antibiotic that inhibits a bacterium MICROAEROPHILIC likes a reduced oxygen concentration MICROTUBULES cell organelles involved in structure. causing adherence to slide. hyphae) IMMUNOASSAY test which identifies antibody in patient based on use of a known antigen. or identifies the antigen based on the use of a known antibody IMViC acronym = indole. methyl red. used for wet mounts .

indicates sensitivity .g. malachite green. pink when oxygenated SEROLOGY the analysis of substances—antigen or antibody--in blood serum SPECIES a subdivision of a genus. for optimizing counts of bacteria in samples POUR PLATE procedure where liquified agar has been poured into a petri dish after being mixed with bacteria PRIMARY DYE the 1st dye used in a differential stain. come-and-go TSB/TSA trypticase soy broth or trypticase soy agar ZONE OF INHIBITION area of no bacterial growth around a chemical on a disc. carbol fuschin RESIDENT FLORA Microbes firmly entrenched in niches in/on the body REVOLVING NOSEPIECE rotating turret attached to the 3 lenses RESAZURIN oxygen indicator in thioglycollate broth.Page 170 PHAGE TYPING test to identify a bacterium based on which known virus infects it PHENOTYPE expression of a gene as a trait PLAQUE destruction of the bacterial lawn by a bacteriophage as the lytic infection progresses PLATE COUNT AGAR variation of nutrient agar. SYMBIOTIC/SYMBIOSIS a relationship between 2 or more organisms THERMAL DEATH TIME minimal amount of time to completely sterilize a specimen at a certain temperature TRANSIENT FLORA Microbes easily dislodged from the body. e. almost identical organisms. crystal violet. a clone SPREAD PLATE procedure where pre-made agar plates have a sample of bacterium placed on top of the agar and spread via a glass rod STREAK PLATE procedure where a bacterial specimen is placed on a pre-made plate and diluted out using flame and multiple sections.

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