Experiment: 1 Bright Field Microscopy Lab Report {pate 22:10. 2011 _ 08.10. 201 Student Name: Lab Section: J-£_ 9.20 Observations and Results Rie spice ‘Trypanosoma gambiense ‘Human Blood (1003 (1000X) (1000) I. Review Questions: Define the following termsExperiment: 1 Bright Field Microscopy I, True/False ‘TAAL. The field of view increases as the magnification increases. . Immersion oil increases magnification power. 3. The round area of light viewed through ocular and objective lens is called field of view. 4, The refractive index of immersion oil is 1.5. 5. Commonly, the magnification of an ocular lens is 10X. ILL Describe the function of the following parts of microscope. Fine adjustment knob: Condenser: A, mersion gil:Experiment: 2 Aseptic Technique and Transfer of Culture Glossary Culture: A bunch or cluster of microorganisms. Pure Culture: A culture that contains only one type of microorganisms. Mixed Culture: A culture that contains more than one type of microorganisms. Medium (plural Media): Any substance, liquid or solid, that facilitates the growth of microorganisms. Inoculum: Microorganisms to be transferred. Inoculate: Transfer of microorganisms from one source to another. Lab ReportExperiment: 2 Aseptic Technique and Transfer of Culture Student Name:,ikiivio. Uroinata Eelibgrate: 08. /2.20)) Lab Section: _T//&_ 4:30 Observations and Results trient agar slant Notiegt brat rient agar deep tube Nutrient agar plate eh gle Re Contamnatens i celta” Soaps IL Review Questions 1k, What is aseptic technique? ee relnique that pounce contami nerhion, 2 pin two tools 5 ‘commonly ws in transferring bacteria? 0 ave A coma To fuolet yeevenet Te Son teed youn | IL. True/FalseExperiment: 2 Aseptic Technique and Transfer of Culture 1, E Itis not important to sterilize loop after a transfer. 2 =A pure culture contains large number of identical daughter cells. 3. =F Streaking bacteria for isolation requires thatthe inoculums be diluted ‘by means ofa loop to individual, separated cells. 4, 1 The second quadrant contains more bacteria than the third quadrant 5._ZT_A broth culture can be used to isolate pure culture.Experiment: 3 Bacterial Motility and Brownian Motion Hanging Drop Method OBJECTIVE ‘To observe and differentiate between true motility exhibited by bacteria and Brownian motion. PRINCIPLE Most bacteria are capable of true motility ‘These microorganisms have appendages called flagella and endoflagella, also called axial filament, (only found "on spirochetes) These locomotor organelles allow bacteria to move independently from one place to another. This allows bacteria to move towards beneficial things like food, oxygen, or light or away from harmful things like toxic chemicals or oxygen (anaerobes). The ability to move in a direction in response to a physical or chemical stimulus is called taxis. Bacteria lacking true motility exhibit a false motion called Brownian movement. This false, non-directional motion is caused when cells are bombarded by water molecules. ‘The hanging drop method used here is a type of wet mount slide preparatici that allows the observation of living, unstained cells in.a fluid medium. EQUIPMENT Materials Depression slide, coverslips, petroleum jelly, tooth picks, Bunsen burner, and wire loop Cultures Nutrient broth cultures of Pseudomonas aeruginosa (18) and Staphylococcus epidermidis (24) pEO CEDURE Using a toothpick, make a ring of petroleum jelly around the cavity on the depression slide. 2. Aseptically, transfer two loopfuls of bacterial culture in the center of the coverslip. 3. With the petroleum jelly side facing, down, position the depression slide ‘over the, coverslip 4, Slowly lower the depression slide, while centering the culture drop in the depression cavity. 5. Gently press the depression slide against the coverslip, just enough to allow the petroleum jelly to make contact. Do not press too hard to spread petroleum jelly to spread outside of the edges of the coverslip. 6. Slowly, lift ‘the coverslip and quickly invert it, The bacterial culture will now be hanging from ‘the coverslip in the depression cavity. 7. Observing under 1000X using immersion oilExperiment: 3 Bacterial Motility and Brownian Motion Hanging Drop Method Lab Report Student Name: Wathiicia. Wrsnita Leblgte Date: 05./2- 20 Lab Section: [7% 4:30 OBSERVATIONS AND RESULTS Record your observations in the following chart. Bacteria ‘True Motility or Brownian Motion Pseudomonas aeruginosa (18) | Staphylococcus epidermidis (24) Review Questions I. Define the following terms: Il. Briefly answer the following questions. 1. In addition to motility, what other cellular activities of living cells can be observed using hanging-drop technique? 2. How would you differentiate between living cells and non-living materials with similar shapes?Experiment: 3 Bacterial Motility and Brownian Motion Hanging Drop Method 's motility play any role IIL. True/False E_1. Only Gram-negative bacteria are motile. “T_2. Number and arrangement of flagella varies from bacterium to bacterium. “>E3. Any living or non-living entity will appear vibrating in a drop of water under the microscope 4. In vitro fertilization can be observed using hanging drop method. E_5. Bacterial motility can be used to differentiate between Gram-positive and Gram- negative bacteria.Experiment: 4 How to Prepare a Bacterial Smear For Staining Lab Report / Student Name: Ld buicua Vrsnita GeldDate: 05.17.2014 Lab Section: - @ 4.30 L Review Questions: UL. True/False F_1.A thick bacterial smear is better than a thin smear for staining, 2. A smear should be heat-fixed when itis partially wet. 3. is not necessary to use water when making a smear from a broth culture. ~ 4. The bacterial smear should be constantly held over flame for atleast 30 seconds to fix it on glass slide. 5. Always use sterilized water for smear preparation.Experiment: 5 Non-Differential Stain Simple Stain Lab Report Student Namechntiicia Urbuia PebteDate: 05 .1¢. Zoll Lab Section: TR _ 9:30 y4 A 2. Bacillus cereus (03) Staphylococcus epidermidis (24) Shape_Rod Shape. Arrangement hows Arrangement (Lele , I. Review Questions: 1. Explain why an acidic dye cannot be used for a sine staining procedure,Experiment: 5 Non-Differential Stain Simple Stain IL. True/False TT _1. Bacteria are negatively charged. 2. Cationic stains are positively charged. E_3. A simple stain canbe used to observe intracellular structures such as ribosomes and nuclear material, £4, Safranin is an example of anionic stain _“F_5. Chromogen can be used to stain bacteria,Experiment: 6 Differential Staining Gram Stain GLOSSARY Anionic or acidic stain: A stain that carries a negative charge. Antimicrobial agent: Any chemical or physical agent that is used to stop the growth of or kill microorganisms. Bacillus (plu. Bacilli): Rod shape or cylindrical shape bacteria, Cationic or basie stain: A stain that has a positive charge. Coccus (plu. Cocei): Round or spherical shape bacterium. Counter or secondary stain: The second and contrasting stain used in a staining procedure. Gram-negative bacteria: Bacteria that retain counter stain, safranin, and appear pink/red. Gram-positive bacteria: Bacteria that retain primary stain, crystal violet, and appear purple. Gram Variable: Gram-positive bacteria that appear pink/red due to old age and over decolorization. Mordant: A chemical agent that binds with the primary color and intensifies it. Primary stain: The first stain used in a staining procedure. ‘Tetrads: Microorganisms growing in groups of four.Experiment: 6 Differential Staining Gram Stain Lab Report Student Name: ls Ubuinta bekte Date: 05./9.20y Lab Section: T-R 9:20 Bacillus cereus ya Grams reaction: Shape: th Arrangement: -Otiaine Suen Basie Escherichia coli (11) Gram’s reaction: Lg, hegiiline Shape: J Arrangement: Duplo bacitle Staphylococcus epidermidis seems UO Gram’s reaction: Varoayein ensign) me =9 tony EY ve stig at ram posts aa 3 on =n iar a Specie tii Aa ‘Surat? 0x09 sR 0 Tino 50 10 = =i emission esp portions of 2A (Perma Saci fo rene Osx Sustain Ts Feu Ein, Aone Sn) has ten ray WCCLS There dala aval ne mado in M2 lve NCCLS equ des ie ele les rough new eons le Sands nd supine ten Users spol ee mod ec eins. Te cute sands ay bd ‘um NCCLS, st atey Rot, Sue 100, Way, A 8087, US ‘clam ng ayo ot anand ein Rss may 0b is pouty ei ayn eet, hes sino aves pubes Macamse ad iy dns F HO, on me ape aN ‘ant pci at pcre Pectin G Sel eae tte Oa SEEM np he ed pn ean sus cise sie pili. snp nc‘ rari sis ase Use ws a a eae Cs fli nein emetic, aso iin, jist, 19 SOE Cond Mp i syExperiment:19 Effect of Antibiotics on Selected Bacteria (KIRBY-BAUER METHOD) Disk Diffusion Technique Lab Report Student Name: hice Votnita Bebite Date: 07,05-20n Lab Section: 7-R 4:50 OBSERVATIONS: Part-A Measure the diameter of clear zone of inhibition around the disks and indicate whether the organisms is resistant ® , intermediate (1) The term intermediate generally means that the result is inconclusive for that drug- organism combination., or susceptible (S). Record your data in the following box: Gram Positive Bacteria ‘Antibiotle Seaureus ‘Susceptibility ‘Susceptibility neers) ens e141 Tetracycline 2g eee F Penicillin 2 = alee a Gentamycin 14 ~ [14 a ‘Vancomycin 5 = 5 Streptomycin 1- = = e sulfisoxazole ay |— S Gram Negative Bacteria Antibiotic E.coli Susceptibility | P. aeruginosa | Susceptibility Zone of Zone of inhibition in Inhibition in R [TI]s RJ I] S Tetracycline 24 = [= oO [El-/= Penicillin } (geass |e Ga) Pel oiler Gentamycin Z a ae tw l-[-[s Vancomycin oO (ns oO Re =-[= Streptomycin © == |—14 Gina sulfisoxazole 2 = a ei @[ =| =Experiment:19 Effect of Antibiotics on Selected Bacteria ~ (KIRBY-BAUER METHOD) Disk Diffusion Technique Acid-fast Bacteria M. smegmatis | Susceptibility Zone of inhibition in mm | [ys Tetracycline 24 =[-16 Penicillin O° 2l-l- ‘Gentamycin 26 --l2 ‘Vancomycin 20 = ([-/?] Streptomycin 20 ail) aL sulfisoxazole 4 =1Is OBSERVATIONS Part-B Record your results in the following chart as “Yes” if synergistic effect (one large zone without any bacterial growth in-between two antibiotic discs) is observed or “No” if additive effect) two separate zones, one around each antibiotic dise with growth of bacteria in-between two antibiotic discs) is observed. Bacteria and antibiotics _| Synergistic effect | Additive effect E. coli Tetracycline and Trimethoprim |_ AQ |e Trimethoprim and Sutfsoxazote |_ ‘T24 | An S. aureus Tetracycline and Trimethoprim aera aie ge ‘Trimethoprim and Sulfisoxazole ea NoExperiment:19 Effect of Antibiotics on Selected Bacteria (KIRBY-BAUER METHOD) Disk Diffusion Technique 1. Review Questions 1, Describe four different major modes of action of antimicrobial chemotherapeutic chemicals and.give three examples of drugs fitting each mode of action 3. List five mechanisms by which microorganisms may resist antimicrobial chemotherapeutic 4 [Can ya titel a reaogn wy a doc gt coe to ie a ids a esate SMALLER zone of inhibition against a pathogen than one that produces a larger zone of inhibition? Yn wot tee funcod hactral resistance 10Experiment:19 Effect of Antibiotics on Selected Bacteria (KIRBY-BAUER METHOD) Disk Diffusion Technique 5. List four genera of microorganisms that produce useful antibiotics, 6. What does “broad spectrum” mean as applied to antibiotics? A Aped Mg pl The eg if Jacly ef 7. Ifyou visit the physician with a bad cold, what antibiotic would you expect the physician to prescribe? Why? 8. Briefly explain why antimicrobial susceptibility testing is often essential in choosing the proper chemotherapeutic agent to use in treating an infectious disease? 9. Briefly explain how you would determine if antibiotic resistance was demonstrated in this experiment? uExperiment:19 Effect of Antibiotics on Selected Bacteria (KIRBY-BAUER METHOD) Disk Diffusion Technique 10. Describe the following terms: a, Synergistic effectExperiment: 20 Effect of Antiseptics and Disinfectants on Selected Bacteria ‘Equipment Bunsen bumer, sterile swabs, centimeter rulers, sterile paper dises, permanent marker, and tweezers. PRECAUTIONS Some of the materials used in this exercise are irritants or even toxic if gotten into the ‘mouth or eyes or if left on the skin too long. Please follow the Instructor's directions for handling these materials safely. PROCEDURE: . Using a permanent marker label the bottom of each plate with the name of bacterium and divide the bottom in 6 sectors and number them 1 through 6 approximately at equal distance from cach other. 2. Preparation of Bacterial Lawn. a. Aseptically remove a sterile swab from sleeve. b. Soak swab with pure culture of any of the five pure cultures. ©. Before removing swab from the culture tube, press it against the GLOSSARY cidal: anti é: antimicrobials that inhibit micr bacteriostatic, fungistatic. inner side of the tube to get rid of excess fluid. 4. Using a sterile cotton swab, gently, evenly spread one bacterial culture on one Muller-Hinton agar plate. (as demonstrated by the instructor), Make sure that (1) entire surface of agar plate is covered, and that the bacterial lawn is not runny. Tilt your plate to see if the bacterial culture runs. If it does run, let it air dry at room temperature for about 5 to 10 minutes before proceeding to the next step. Repeat step 2 a, b, o, and to prepare bacterial lawns of other bacteria. 4. Using sterile forceps, impregnate a sterile disc with the first antiseptics or disinfectants ‘you wish to evaluate and place the dise on ihe appropriate number of the plate, Place the disc near the edge of the Petri dish, but without touching it. Gently press the di down to ensure firm contact with medium. 5. Repeat step 4 for each Petri dish for each bacterial culture you are using. 6. Repeat steps £4 and 5 for each antiseptic / and disinfectant you are using. 7. Secure all plates with masking tape and incubate them upside down at 37°C until next lab period. jcrobials that kill microbes, e.g., bacteriocidal, fungicidal antimicrobials that kill microbes by_lysing them, e.g., bacteriolytic.Experiment: 20 Effect of Antiseptics and Disinfectants on Selected Bacteria Lab Report , D Student Name: ti uo Veninike tdite Date: 07.07.2011 Lab Section: R 4-30 Rate the efficacy of each antiseptic/disinfectant against the test bacteria using a scale of 0 = no effect; 1+= little effective; 2+= somewhat effective; 3+=average; 4+=Effective; and 5+ = very effective. Record your data in the following box: “Antiseptic / ‘Gram-positive ‘Gram-negative ‘Acid-fast Disinfectant ‘S.aureus |S. pyogenes E.coli] P.aeruginosa | M. smegmatis 70% Alcohol Ze Te T+ iia Tincture of fodine Ay 4¥ 3+ ik Crpstal violet ¥ o oO aCe Tisterine © o. © © Ll, Ar O | © 4 Hydrogen peroxide | A+ ar | 3h + ‘After recording your data, discard the plates in the biohazard container. Review questions:Experiment: 20 Effect of Antiseptics and Disinfectants on Selected Bacteria 3. According to your results, which chemical is the most effective? On what do you base this conclusion? H 22 Wypeauece it hel a mani ime of bali lotions 4, Which chemical was not effective against a gram-positive bacterium? Against a gram-negative bacterium? How does this influence where yoy use these chemicals? 5, How could you determine whether the bacteria in a zone of inhibition were killed or just inhibited? Jorroving the dives anol mcybactunn if bh eeraee ate The actinic hs Wad La ih f a. AntiseptieExperiment: 21 Bacterial Conjugation Lab Report . Student Nine: ity ini le Date:_ 07.07. 201) Lab Section: 7, 30. Observations and Results I. Record your results in the following chart. Growth E. coli culture | Present or absent Mix_ nbatart F- hacut Bir Obrevet Reading the results: Plate-1: F-E,coli (auxotrophic, thr-, leu-, thi-, and str-r) Observations There should be no growth. Although the bacterium is streptomycin resistant but it does not have the genes to synthesize essential amino acids threonine and lucine. Plate-2: Hfr E. coli (prototrophic, wild type, str-s) Observations There should be no growth. Although bacterium is capable of synthesizing thiamine, but it is streptomycin sensitive.tts growth will be inhibited by the presence of streptomycin in the medium. Plate-3: Mixture of two cultures Observations Growth should occur. F- bacterium is streptomycin resistant, but is unable to synthesize threonine and leucine. After short incubation period, F- will acquire threonine and leucine genes from Hifr bacterium.Experiment: 21 Bacterial Conjugation IL. Review Questions 1. Briefly explain the importance of bacterial genetic exchange in nature? 2, How does the exchange of genetic information help bacteria survive? Can you give an example? 4. Are the original bacteria provided in this lab resistant to streptomycin? How will you be able to prove this? 5. How will you be able to detect the bacteria that have successfully taken in the plasmid (been transformed)?Experiment: 21 Bacterial Conjugation IIL, Define the following terms: «eas the 2oves b. Franatucion £. PlasmidExperiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate Citrate Test inoculum down through the butt, then pull the needle out and streak up the slant (Do a. Inoculate the Simmon’s citrate agar tubes NOT take another inoculum to do the using an inoculating NEEDLE. stab the slant) b. Incubate al slants at 37°C. until next lab periodExperiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate Lab Report : Student Name: Sftrivia Vedmike Bele Date: 0% /2 2.01 Lab Section: T-@ 430 Observations and Results: Indole test Add the five drops of Kovac's reagent to detect the presence of indole produced. Within just a few seconds you can see the pink color of indole presence. Methyl Red and Voges-Proskauer tests Pour half of the MRVP broth into a clean empty test tube: Use one tube to perform MR test in the other tube to perform VP test. Methyl Red Test Add 10 drops of Methyl red reagent to the tube and within just a few seconds you can see the red- pink color indicating the presence of acid. Voges-Proskauer Test ‘The reagents MUST be added in the correct order, in the correct amounts, and the tube must sit undisturbed, and open to the air (no cap) for atleast 30 minutes_as the light pink color intensifies at the top of the tube (the reagents react with acetoin). DO NOT shake the tube after sitting it down for the waiting period Citrate Test No reagents need to be added after incubation. Just observe the citrate for a change from green to Prussian blue. The alkaline by-products of citrate use will cause the pH indicator to turn Prussian blue. Blue is considered positive for this test. Remove all marks from the tubes and place them in the designated area.Experiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate Record your results in the following chart. Bacteria '[Indol test | MR test | VP test | Citrate test +or- +or- +or- +or~- = coli (11) Heme) + ed) = Gello) — Green) erogenes (09) | (ela) Helin) F (ied F Colne) Paar [tonal Ftved)| Gedy EF Cetue)| REVIEW QUESTIONS 1. Complete the following chart Bacteria Indol test_| MR test VP test Citrate test End End End End product(s) | product(s) product (s) product (s) E. coli (11) Ende [Add = er. E. aerogenes (09) = No Aad No feid. |Sooliam Biathavet P. vulgaris (17) Froel | Ad = Bodiam be cartowe: 1. What is the substance found in Simmon’s Citrate Agar that is the sole carbon source of the media? Ao tbaate 2.71 /me that would be present in bacteria that could transport citrate would be? tow cake permease 3. If) i aay cing tums Prussian blue what is the pH of the media?Experiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate 4, Citrate is a part of a battery of tests, Fill in the Chart 6. What is the pH of an un-inoculated Simmon’s Citrate Agar Plate? 7. Is it possible for an organism to be Methyl red and Voges-Proskauer positive? Explain your answer.Experiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate 8, Escherichia coli and Enterobacter aerogenes look very similar in a microscope. What testing procedures would you use to tell them apart? EMVic Teak Self-test lo 1. The Voges-Proskauer test is used to differentiate member of the: @Spirochetes (b) Enterobacteriaceae (@)Endospore forming (@Gram-positive cocci (©) Gram-positive rods 2. Place a check by the substances that are found in MR/VP Broth phenol red Iron. cysteine lactose starch__ glucose peptone phosphate buffer acetoin 3. What are the chemicals that compose Barrit’s A reagent? Olpho - waphtoe 2 4, What are the chemicals that compose Barritt’s B reagent? Potastium atlydeecs oe 5A burgundy color at the top of a VP testis: (a) a negative reaction and due to the reaction of KOH and alpha naphthol a positive reaction due to KOH reacting with a product produced by the bacteria and the ‘media (©) anegative test and due to reaction of acetoin and KOH (@ a positive test due to the reaction of peptone and the Barritt’s A and BExperiment: 22 Biochemical Test IMViC Indole, Methyl Red, Voges-Proskauer, and Citrate 6. Give the significant ingredient(s) of MR/VP broth. a oto Pe ptone 7. For all of the following test give the positive and negative colors or reactions: ‘Voges-Proskauer Test Methyl Red 8. What chemical substance is produced in Methyl Red and if present gives a positive reaction? XJtalole Acud 9.What chemical substance is produced in Voges-Proskauer test and if present gives a positive reaction? Non frewl evel paodauc Qeetorn lola nected 10. The purpose of the citrate in the Simmon’s citrate medium is to determine ifthe organism can used citrate as the sole COW.| ‘+ source. 11. The end product identified in the" VP test is a neutral compound called _QCé houExperiment: 24 Biochemical Test Carbohydrate Fermentation OBJECTIVE This test is used to determine the ability of an organism to ferment various simple carbohydrates (sugars) PRINCIPLE Carbohydrates are complex chemical substrates which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen, and oxygen. Facultative anaerobic and anaerobic bacteria are capable of fermentation. In this anaerobic process, the final electron acceptor is an organic molecule. Each medium has a single fermentable carbohydrate added to a peptone medium. Phenol red is also added as a pH indicator. A small tube (Durham tube) is inverted and placed in each larger test tube of liquid medium, The inverted tube is able to trap any gas products. ‘The indicator, phenol red will turn yellow below pH 6.8 (acidic) and a darker pinkish- red above pH 7.4 (alkaline). If the organism metabolizes the carbohydrate, subsequent acid production will result in lowered pH. If the organism does not ferment the carbohydrate, the pH may remain neutral. If the organism does not ferment the carbohydrate and also utilizes the peptone, accumulation of the ammonia as a degradation product will raise the pH. MATERIALS Equipment: Bunsen bumer and wire loop Cultures: Nutrient broth cultures of: Pseudomonas aeruginosa (18) Escherichia coli (11), and Staphylococcus aureus (23). Media: Per pair 3 tubes each of: Phenol red lactose broth, phenol red glucose/dextrose broth, and phenol red sucrose broth. PROCEDURE ALL carbohydrate tubes have color coded caps. Red = Dextrose/Glucose Blue = Lactose Green = Suerose Make sure not to shake broth tube as it may cause entrapment of air bubbles in the Durham tube and may give you a FALSE POSITIVE result for GAS. 1. Using a permanent marker, label each tube with the name/number of the organisms.2. Aseptically, Inoculate one phenol red lactose broth tube, one phenol red glucose/dextrose broth, and one phenol red sucrose tube with E. coli. 3. Aseptically, Inoculate one phenol red lactose broth tube, one phenol red glucose/dextrose broth, and one phenol red sucrose tube with S, aureus. 4. Aseptically, Inoculate one phenol red lactose broth tube, one phenol red glucose/dextrose broth, and one phenol red sucrose tube with p. aeruginosa. 5. Incubate all tubes at 37°C until next lab period.Experiment: 24 Biochemical Test Carbohydrate Fermentation Lab Report Student Name: fAtucca Limba Btpate: 0F.19. 201 Lab Section:_J-@. 2:20 OBSERVATIONS AND RESULTS: Reading the Test Results Acid: (yellow) Acid production produces a color change from red to yellow, indicating that the organism is capable of metabotizing the sugar in the tube. Acid, Gas: (yellow plus gas bubble) Fermentation of the sugar is indicated by a color change to yellow. Gas is trapped in the Durham tube, replacing the medium in the tube. A bubble indicates gas production, Negative: Negative fermentation can be indicated two ways: 1. No color change in the tube means that the sugar was not utilized by the organism. 2. Color change to a dark, pinkish-red: this darker color indicates alkaline or basic metabolic products ‘hich is due to the utilization of the proteins, rather than the sugar. If the tube is read within 48 hours, the darker red color would be an indication of negative fermentation; although the result is usually recorded as alkaline, A=acid, G= gas, AG= acid and gas Organism Glucose/dextrose ‘Sucrose Fermentation | Lactose Fermentation Fermentation(red eap) (green cap) (blue cap) ‘Acid [Gas] Both | Acid | Gas] Both | Acid | Gas | _ Both Acid and Acid and ‘Acid and (a) | @ | Gas (A) | (a) | G | Gas AG | (A) | G | Gas (AG) E. coli (11) a emer ee lo Clhansey AG aureus (23) A= = A [= area An eee Plaeruginosa G3) | — | — = |= ==Experiment: 24 Biochemical Test Carbohydrate Fermentation REVIEW QUESTIONS 1, What are the three groups of carbohydrates? Give an example of each. 2. Which of the above groups does starch belong to? Pelt sacelericles 3. Explain what happened in a test tube of Phenol Red Lactose Broth if: a. there is a bubble in the Durham tube \ncbrcaten He presence of Sau b. acolor of yellow Nirdicaitén Awol Produotion 4, Give the color for Rud a. an un-inoculated phenol red broth one herd b. abroth at pH3 ‘Dark Prrak Rode. a broth at pH 8.4 Real d. a broth at pH 7.0 ot 7.2 5, What substance in the Phenol Red Broth is acted upon to produce: 4) an alkaline condition Rrotein ) an acid condition —Canloobydrale ) a gas bubble trapped in the Durham tube_—Coan looky ohate324 Biochemical Test Carbohydrate Fermentation Experiment Select the best possible answer. 6. Fermentation normally produces a(n) a. alkaline environment () acidic environment ‘e.neutral environment 7. List the ingredient in the broth that contributes to an alkaline condition inthe broth if b. phenol red peptone @. none of the above 8. Explain why the Phenol Red Broth would need more carbohydrate than protein, if the broth ‘would be incubated for a longer period of time. Qui np Beg ly | Lopich fo. bra Bie eet al Phar CaskExperiment: 25 Biochemical Test Triple Sugar Iron Report Student Name divalent: OF.14 201 Lab Section: 1-~ AZO OBSERVATION AND RESULTS Record your results in the following chart. Bacteria Sugar Fermentation TES Production a ‘i ee a 2 ca Rat i |e E.coli (11) ¥ Vv ¥ Te Om Ss we sen Te “Pyle Bueete| ss aq [be LY | Suatate z Pate | & ES room Ty el Tet Ans | oe INTERPRETATION OF RESULTS If an organism ferments glucose only, the entire tube tums yellow due to the effect of the acid produced on phenol red. Because there is @ minimal amount of glucose present in the tube, the organism quickly exhausts it and begins oxidizing amino acids for energy. Ammonia is thus produced and the pH rises. Within 24 hours the phenol red indicator reverts to its original red color on the slant. Because TSVKIA media is poured as a deep slant, the butt has limited oxygen and bacteria are unable to oxidize amino acids there. The butt thus remains yellow. Ifan organism can ferment lactose and/or sucrose, the butt and slant will turn yellow (as they do from glucose fermentation). However, they remain yellow for at least 48 hours because of the high level of acid products produced from the abundant sugar(s) If the gas being produced is hydrogen sulfide (HS), it reacts with the ferrous sulfate and precipitates out as a black precipitate (ferric sulfide) in the butt. Organisms producing large amounts of hydrogen sulfide (e.g. Salmonella and Proteus) may produce so much black precipitate that it masks the yellow (acid) color of the butExperiment: 25 Biochemical Test Triple Sugar Iron Report Student Name divalent: OF.14 201 Lab Section: 1-~ AZO OBSERVATION AND RESULTS Record your results in the following chart. Bacteria Sugar Fermentation TES Production a ‘i ee a 2 ca Rat i |e E.coli (11) ¥ Vv ¥ Te Om Ss we sen Te “Pyle Bueete| ss aq [be LY | Suatate z Pate | & ES room Ty el Tet Ans | oe INTERPRETATION OF RESULTS If an organism ferments glucose only, the entire tube tums yellow due to the effect of the acid produced on phenol red. Because there is @ minimal amount of glucose present in the tube, the organism quickly exhausts it and begins oxidizing amino acids for energy. Ammonia is thus produced and the pH rises. Within 24 hours the phenol red indicator reverts to its original red color on the slant. Because TSVKIA media is poured as a deep slant, the butt has limited oxygen and bacteria are unable to oxidize amino acids there. The butt thus remains yellow. Ifan organism can ferment lactose and/or sucrose, the butt and slant will turn yellow (as they do from glucose fermentation). However, they remain yellow for at least 48 hours because of the high level of acid products produced from the abundant sugar(s) If the gas being produced is hydrogen sulfide (HS), it reacts with the ferrous sulfate and precipitates out as a black precipitate (ferric sulfide) in the butt. Organisms producing large amounts of hydrogen sulfide (e.g. Salmonella and Proteus) may produce so much black precipitate that it masks the yellow (acid) color of the butExperiment: 25 Biochemical Test Triple Sugar Iron TYPICAL REACTIONS OF SELECTED ENTERICBACTERIA + [one of "para-colon” group + |Causes GU infections, highly motile ~ [Peano indebted pons ooscomia) opportunistic pathogen of GU 2. What would the medium look like if the bacterium has alkaline slant and acidic butt with Com 2 + 5 p ellos with Kosa lohen 3, What is the color of the test tube if: a. glucose fermented Celle) Butt | peed lant . hydrogen sulfide present Black. Decipitar wn Real c. uninoculated TST 4. glucose, sucrose and lactose fermented VeLlous Meet anal aban peptone catabolized Rel butt anol slantExperiment: 25 Biochemical Test Triple Sugar Iron 4. 5. Explain why TSI tests should be observed within 18 to 24 hours. Cite the source of your answer. 6. Could reliable results be obtained if TSI agar were inoculated with a sample directly from a fecal suspension? Why?Experiment: 26 Biochemical Test Extra-cellular Enzymes Lab Report Student Nam WPetniv, a thisnite Bolte Date: 6% 21.200 Lab Section: T= 2 4°20 OBSERVATIONS AND RESULTS Test for starch hydrolysis. Flood the starch agar plate with Gram’s iodine solution. Allow to react for two minutes. The plate should turn purple, except where the starch has been hydrolysed, usually around the area of growth. No purple color, a clear zone around the bacterial growth, indicates area of the plate where starch has been hydrolyzed. The readings must be made within three minutes because the starch iodine complex will fade rapidly. Test for casein hydrolysis. Examine the milk agar plates for evidence of clearing around the colonies. This zone is where the bacteria have hydrolyzed the casein in the milk agar plates.( Milk agar plates start off opaque). A clear zone around the bacterial growth indicates casein hydrolysis . Test for gelatin hydrolysis Place the gelatin tubes in the refrigerator for at 45 minutes before examining for gelatinase activity. ‘Tubes that remain liquid after refrigeration are positive for gelatinase. Record your observations in the following chart. Bacteria Clear zone around bacterial | Gelatin after | Name of growth Refrigeration | Hydrolytic Starch Milk Agar Enzyme Present or Absent _| Present or Absent | Liquid or Solid B. cereus (03) + Ca + 3 E. coli (11) as = = NI P. aeruginosa (18) = ¥ + Lectiwaad | Coss S. aureus (23) i eS = Cascinerst Review QuestionsExperiment: 26 Biochemical Test Extra-cellular Enzymes 2. What is casein, where is it found, and what results in the laborator U 0 3. Complete the following chart. ‘Substrate | Enzyme | End-product | Regent (fany) Starch |STaneh | Avy laae tout | Todine | last | Gelatinase [Aiminohud ‘asein 5 4, What is iodine used to test for? Storch. Hy deadar nt 5. What is the common suffix found on the names of almost all enzymes? ~ OAL,Experiment: 27 Immunological/Serological Test Agglutination Test Lab Report Student Name: Nes ate:_OF 21. 201 Lab Section: T= iz OBSERVATIONS AND RESULTS Record your observations in the following chart. Agglutination Negative Control Patient Serum. Positive Control Present or Absent. Present or Absent Present or Absent = ee Based on your results: Does the patient have the disease he/she tested for? Briefly explain your angwer. he the olescatt Gecause how Seu, Week el antl tb REVIEW QUESTIONS sg 1. Define the following: a. Antigen wtulstomee thot stair an wre Pespoase . AntibodyExperiment: 28 Lab Report Nitrate Reduction Test Student Name: bitin Ved Suite BdMate: 07.26.20 Lab Section: T= 4:30 OBSERVATIONS AND RESULTS Look for Nz gas first before adding reagents. © Add 6 drops of nitrite reagent A. * Add the same number of drops of nitrite reagent B. You should see a reaction within a minute or less. Ifyou have not seen either nitrite or N2 gas, you need to add a pinch of zine crystals. Record your results in the following chart. Color after | Nitrate Reduction ‘Nitrate Reduction sdaing | yesrnoorcannot | Cionatet | yes or noltyes, reagents A | (tell Ifyes, name ting 2 name the end and B the end product Pe product Acfuceats() No Colm [Govanot Tell] Real No E. coli (11) Rel eaQNoo)| N/A NK Pacruginosa(t8) [Nv Cole [Cannot Tell | No Coler_| tea Amon INTERPRETATION Nitrate reduction is detected with the Griess Llosvay reagents, sulfanilic acid and alpha- naphthylamine. ‘Sulfanilic acid (Nitrate reagent A) is added to the incubation mixture and forms a complex (nitrite- sulfanitic acid) with any nitrite present in the medium. When alpha-naphthylamine (Nitrate reagent B) is added to the incubated medium, a red precipitate (prontosil) will be formed with any nifrite-sulfanilic acid complex present in the medium. ‘An organism may be reported as nitrate- positive if-ared color develops in the medium after Nitrate reagents A and B are added to the medium, indicating that the organism has reduced nitrate to nitrite. ‘The absence of a red color after the addition of both reagents does not automatically mean that the organism is unable to teduce nitrate. Strains ‘may have reduced the nitrate to nitrite, and then reduced the nitrite completely to nitrogenous gases which are not detected when Nitrate reagents A and B are added to the medium. If the ‘medium does not change color after the addition of sulfanilic acid and alpha-naphthylamine, a small amount ("knife point") of zine dust is added to the incubated medium. The zinc dust will catalyze the reduction of nitrate to nitrite chemically. Thus, if the nitrate has not been reduced by the organisms, i.e., they are nitrate- negative, it will be reduced by the zine dust and a red color will develop in the incubated medium within 15 min, If no color develops in the incubated medium after the addition of zinc dust, the organisms have not only reduced nitrate to nitrite, but have reduced nitrite to nitrogenous gases; these organisms are also nitrate-positiv.Experiment: 28 Nitrate Reduction Test ae ee ere zi | "Color after [Color after adding | REACTION | Nags gguinp reagent ~ (not added) _ REVIEW QUESTIONS 1, Name the 2 major end produets of nitrate reduction. Noite ard Oimmenia 2. Why add zinc powder? When do you add it? 4. Wnt) reduction sa berbic pathway or an anaerobic pathway?Experiment: 29 Normal Flora of The Human Skin Lab Report Student Name: Sti sin Urinike Bllste Date: 62.26. 2011 Lab Section:_t-2 4'30 OBSERVATIONS AND RESULTS Record your observations in the following chart, Blood Agar Mannitol Salt Sabouraud Agar ‘Agar Beta ‘Alpha | Gamma Mannitol Yeast | Mold Hemolysis | Hemolysis | Hemolysis | Fermentation | Present | Present Present or | Presentor | Presentor | (D)Presentifplateis | or | or absent absent | abseat (no _ | yellow and has absent | absent Gtthereis | (ifthereis | zone/hemolysis | golden yellow growth slear greenish ) 6S. aureus) zonelcomplete | zome/partial Or Iysisof RBC) | ysis of @) absent i plate is RBC) stil pink and has white growth (S. epidermidis) = = +J/— +/-| +E Present = + Absent = - Review Questions: 1. How many types of staphylococci you see on your Mannitol agar plate? What is the approximate ratio of pathogenic ang non-pathogenic staphylococci? Occ re presenge of large quantities of S. aureus oon an individual's skin?Experiment: 29 Normal Flora of The Human Skin 4. What ato medial digi of complep ack of growth onary ofthe plates? 5. How would you differentigte between yeast and mold? % worn Mw M1 pest 6. What are the roles of mannitol and salt in the mannitol salt agar? Em ely Cobian ion oflel is. EE Sima til of PF le trea < leuund) ood ~ Jeni Afhect lynolrense =™ TRS Boa Sse 8. Can you explain the difference in prevalence of S. aureus between the general public and hospital staff? a Key p froma Aetwavivaly lied ley f tg nel ort cede stead chExperiment: 29 Normal Flora of The Human Skin 11. Describe the three types of hemolysis, mae? Lat Frcs Slew (an a clean oa) anticl te Ooe Al vhe hamolfie 1a partial bgtaa of RE : 13. Can a healthy person have a throat containing a lot of beta-hemolytic streptococci? Explain, 15. Compare your flora with those of your neighbors. Is there a difference and if so what, does this mean?Experiment: 30 OBJECTIVES To detect the presence of enzyme catalase synthesized by selected aerobic and facultative anaerobic bacteria. PRINCIPLE Living cells go through cellular respiration to generate energy in the form of ATP to carry out various cellular activities. This ‘energy is produced in a series of oxidation, loss of hydrogen ions and its electrons, and reduction, gain of hydrogen ions and its electrons reactions. Hydrogen ions and its electrons released during cellular respiration cannot remain free in the cell (free radicals) and must be removed immediately. Many aerobic and facultative anaerobic bacteria synthesize an enzyme called catalase which initiates the breakdown of toxic form of oxygen such as hydrogen peroxide (H;0;) into water (H,0) and free oxygen (0;). H,0; + Catalase ———> 21,0 + 02 During the normal process of aerobic respiration (Glycolysis, transition reaction, and the Kreb’s cycle) hydrogen ions (H’) are given off and picked-up by NAD and FAD and taken to. the electron transport chain to be utilized as source of energy to run electron transport chain, At the conclusion of the electron transport chain, these hydrogen ions. must be removed by the cell. A. enzyme called cytochromes oxidase combines these hydrogen ions with oxygen to form water (H,0). During the process, energy is given off and is trapped and stored in ATP. Water is then a harmless end product. Some cytochromes in the electron transport system, however, form toxic hydrogen peroxide (H;O,) instead of water Catalase Test and this must be removed. Most bacteria are catalase-positive; however, certain genera (groups) that don't carry out aerobic respiration, such as_Streptococeus, Lactobacillus, Mycoplasma, and Clostridium, are catalase-negative, Bacteria that are catalase-negative but are able to grow in the presence of oxygen have other enzymes like peroxidase, superoxide dismutase, and superoxidase, to remove toxic forms of oxygen such as superoxide and O2-,an unstable form of oxygen. MATERIALS Equipment Bunsen bumer and a permanent marker. Cultures Trypticase Soy agar slant cultures of Micrococcus luteus, Staphylococcus aureus, and Streptococcus lacti Reagents 3% hydrogen peroxide (H:02) PRECAUTIONS 1, Do not use media containing blood because red blood cells contain catalase. 2. Use a fresh bottle of reagent PROCEDURE Add a few drops of 3% hydrogen peroxide to cach culture and look for foaming due to the release of oxygen as a result of hydrogen peroxide breakdown,Experiment: 30 Catalase Test Lab Report Student Nene ie eee aoe OF .28.201 Lab Section: +-2 4.30 Observations and results Record your observations in the following chart, Bacteria Foaming or Catalase No Foaming Present or 5, Absent M. luteus (15) Foams. resent S. aureus (23) Foanivd cae S. lactis (26) No Foatine [Aheodk J d I. Review Questions: 1, State the function of the enzyme catalase and describe a method of testing for catalase activity. 2. Explain how catalabe-negative bacteria are capable’ of growing in the presence of IL Define the following terms: a. Cellular respirationExperiment: 30 Catalase Test >. Oxidation cs of ebeclims anol Hey eosen Lona cc. ReductionExperiment: 31 Qualitative Analysis of Milk Methylene Blue Reduction Test Teucocytes affect the reduction time ‘materially. Light speeds-up reduction and therefore the tests should be kept covered. ‘The concentration of the dye should be uniform as an increased concentration Iengthens the time of reduction, Increasing the incubation temperature augments the activity of the bacteria and therefore shortens the reduction time.Experiment: 31 Qualitative Analysis of Milk Methylene Blue Reduction Test Lab Report Student Name: Lets fsinrfa Bolte Date: 03.28. 200 Lab Section: 3:30 - Record your observations in the following chart. Pasteurized Milk Raw Milk Reduction Time Quality of Milk. 00. | eaten Sen. Cot Review Questions 1. Explain why milk sours if eft in the refrigerator passed freshness date? sep tae ates row He vf haaliveo, urhere 2. List 4 major sources of bacterial contamination of milk.