ENVE 202 LABORATORY REPORT

EXPERIMENT-5
Submitted by: Group-1.6 Sarp ELEB Nazl B. DOAN Prl T. ERDEM Ghazal KHODKAR

Submitted to: Firdes YENLMEZ

03.04.2013

1. PURPOSE The aim of this experiment is observation of effects of disinfectant, UV light and different antibiotics on bacterial growth. Another purpose of the experiment is to learn part of microscope and how to use the microscope by calibration method. Finally, the observation of microorganisms like bacteria, yeast and fungi is purposed by using microscope.

2. PROCEDURE

Effect of Disinfectant on Bacterial Growth: Four nutrient agar plates are taken and E.coli culture is spread on their surfaces in order to get a dense culture. Four test tubes, containing 9 mL sterile water, are taken and 1 mL of concentrated disinfectant, Zefirol is mixed with 9 mL water of one of the tubes in order to get 10-1 dilution Then 10 , 10 ,10-4 dilutions are prepared by using first tube (1mL zefirol+9mL water). Four paper disks are taken, soaked in each dilution tube and placed in the middle of each agar plate that were prepared in the beginning The agar plates are put on the 35 C incubator, and the growth is checked the day.

Effect of Antibiotics on Bacterial Growth: 0.2 mL of bacteria culture is spread onto an agar plates. Three paper disks are taken and each one is soaked into one of the antibiotics. Then paper disks are put on the same agar plate that was prepared in the beginning, away from each other. The plates are then put into the 35 C incubator, and the growth is checked the next day.

Effect of UV Light on Bacterial Growth: E.coli is spread onto an agar plate. A line that splits the plate into two is drawn and half of it is covered with paper. After that, the plate is placed right under the UV light source and exposed to it. This procedure is followed by each group, but for different durations. We did for 30 minutes. Finally, the plate is put into the 35 C incubator and the growth is checked the next day. 1

Microscope Three lams and lamels are taken. A drop of water is dripped on each of them so as to place the microorganism samples. After a loop full of microorganisms is placed, lamels are put on lams with an angle of 45.

Microscope Measurement Calibration is done by deciding on how many milimeters in stage micrometer equals to how many lines in the graduated eye piece. Pore size is measured by the method and with objective magnification.

3. THEORY There are two main ways of microbial control which are (Madigan et al., 2012): 1. Physical control: Heating, radiation (UV, ionizing, radiation particles), filtration (Madigan et al., 2012) 2. Chemical control: Sterilants, disinfectants, sanitizers, antiseptics, antibiotics (Madigan et al., 2012) One of the physical control methods is using ultraviolet radiation. UV radiation having a wavelength between 220 and 300 nm, has enough energy to alter or break the DNA of the exposed of organism. This method is useful for disinfecting surfaces, air and materials like water which do not absorb the UV light. Yet, UV light cannot penetrate solid, opaque and light observing surfaces (Madigan et al., 2012). One of the chemical control methods is using disinfectants. Chemical agents that kill microorganisms (not necessarily endospores) and which are used on inanimate objects such as floors, tables etc. are called disinfectants (Madigan et al., 2012). Properties of an ideal disinfectant are: Having a broad spectrum (URL 7) Acting fast (URL 7) Not being affected by environmental conditions (being active beside organic matters or other substances) (URL 7) Being non-toxic and non-irritating (URL 7) Not being corrosive (URL 7) Being easy to use (URL 7) Being odorless or having a pleasant odor (URL 7) Being economical (URL 7) Being water soluble and forming stable solutions (URL 7) Being a good cleaning agent (URL 7) Being non-flammable (URL 7) 2

Factors that affect disinfectant activity are: Number of microorganisms (URL 3) Location of microorganisms (concerning hard-to-reach parts of objects) (URL 3) Microorganism resistance (URL 3) Disinfectant concentration (URL 3) Disinfectant strength (URL 3) Temperature (URL 3) pH (URL 3) Relative humidity (URL 3) Water hardness (URL 3) Presence of organic and inorganic matter (URL 3) Exposure period (URL 3) Disinfectants function by: Clumping cell proteins (including enzymes) (URL 10) Oxidizing proteins, lipids and carbohydrates (URL 10) Penetration and disruption of cell wall (URL 10)

Antibiotics are the substances that are used to eliminate microorganisms which can be ingested and injected to treat diseases (Tortora et al., 2013). Table-1: Selected antibiotics and their spectrums and effective substances Name of antibiotic Spectrum Effective substance Augmentin Bacillus anthracis, Amoxicillin and Clavulanic Corynebacterium species, acid (URL 2) Enterococcus faecalis, Listeria monocytogenes, Nocardia asteroides, Streptococcus, Clostridium, Peptococcus, Bordetella pertussis, Brucella species, Gardnerella vaginalis, Legionella species, Neisseria, Pasteurella, Proteus, Salmonella, Shigella, Vibrio cholerae, Yersinia enterocolitica, Bacteroides species, Fusobacterium, Borrelia burgdorferi, Chlamydiae, Leptospira icterohaemorrhagiae, Treponema pallidum (URL 2) Equizolin Staphylococcus aureus Cefazolin sodium (URL 3) 3

Zinnat

(including -lactamaseproducing strains), Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and other strains of streptococci, Streptococcus pneumonia, Escherichia coli, Proteus mirabilis (URL 1) Haemophilus influenzea, Cefuroxime axetil (URL 11) Haemophilus parainfluenzae, Moraxella (Branhamella) catarrhalis, Neisseria gonorrhoeae, Escherichia coli, Klebsiella strains, Proteus mirabilis, Providencia strains, Proteus rettgeri, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Borrelia burgdorferi, Propionibacterium strains, gram-negative bacilli, grampositivi bacilli, grampositive and gram-negative cocci (URL 11)

Visualization of microorganisms requires microscopes. Light microscopes will be examined because of this reason (Madigan et al., 2012). A light microscope has eye-pieces, ocular lenses, an objective lens, a stage, focusing knobs, a light source and a condenser. By the help of visible light and the lenses, the specimens are magnified. Total magnification of a light microscope is the product of objective and ocular lenses and the upper magnification limit of light microscopes is approximately 2000x. Resolution, which is described as the ability to distinguish two adjacent points as separate and dependent on lights wavelength and numerical aperture, does not improve after 2000 times magnification. And the formula for the diameter of the smallest object resolvable by any lens is 0.5/numerical aperture. Resolution of microscopes can be improved by the use of immersion oils (Madigan et al., 2012). An ocular micrometer is a measuring device with indefinite units (since the unit varies with magnification) that is placed inside the microscope (URL 8). A stage micrometer is another device that is put on the stage and has definite measures on it. To be able to do length measurement with a microscope we have to use ocular micrometers, since both the 4

specimen and the stage micrometer cannot be placed on the stage at once, even though stage micrometers are very accurate. So ocular micrometers are calibrated by the help of stage micrometers (URL 12). 4. DATA ANALYSIS & CALCULATIONS Effect of Disinfectant on Bacterial Growth Table-2: Effect of disinfectant Zefirol at different concentration values Dilution Ratio Death Rate -1 10 +++ -2 10 ++ -3 10 + -4 10 -

Effect of Antibiotics on Bacterial Growth Table-3: Antibiotics death rate Antibiotic Augmentin Zinnat Equizolin Where; +++: good growth ++: fair growth +: poor growth - : no growth Effect of UV Light on Bacterial Growth Table-4: Exposure time and death percentage Time (min) Death (%) 0 0 5 20 15 50 20 65 30 95 Death Rate + ++ +++

Death percentage (%) vs Exposure time (min)

120

y = 3.1228x R = 0.998

Death Percentage (%)

100 80 60 40 20 0 0 0; 0 5 5; 20 10 15 20 25 30 20; 65 15; 50

30; 95

death

35

Exposure Time (min)

According to the Graph.1 time required for nearly all of the bacterial cells to be killed is approximately 30 minutes.

Microscopic Measurement Total magnification = Eye-piece magnification * Objective magnification Eye-piece magnification = 6.3 Objective magnification = 10 So; Total magnification = 6.3 * 10 = 63 In the microscope we observed the pore size as 10 units. 10 units 1.45 mm 1 units x ============== X = 0.145 mm

Figure-1: E.coli view from microscope (URL 6)

Figure-2: Yeast view from microscope (URL 6)

Figure-3: Fungi view from microscope (URL 4)

Figure-4: Algae view from microscope (URL 9)

Figure-5: Protozoa view from microscope (URL 13)

5. DISCUSSION & CONCLUSION In conclusion, the experiments, which have been performed last week, the effects of disinfection, antibiotics and light (UV) on the microbial growth have been observed. In addition to these, the microscopic scales and application of them have been shown. Disinfection is a kind of microorganism removal process. Usually it is mixed with the term sterilization. Actually they resemble but the main difference is that the sterilization is the removal of all microorganisms (Tortora et al.). However, disinfection is the removal of a specific type of microorganism. In the first experiment, which has indicated the effect of disinfectants on microbial growth, Zefirol is used as a disinfectant. In this experiment, the effect of Zefirol is measured for several dilutions. There are four samples which have different dilution factors. According to table 1, it can be said that as the dilution factor increases, death zone around the paper disc gets larger. From the results, it can be said that effectiveness of a disinfectant depends on the dilution ratio. The importance of disinfection in environmental engineering processes is to obtain isolate cultures. There is need for isolate cultures in environmental engineering in biological treatment processes (Tortora et al.) Another microorganism removing object is antibiotics. Antibiotics are the chemicals produced by some kind of bacteria and fungi against other microorganisms (Tortora et al.). In the second experiment, three different antibiotics are used: Augmentin, Equizolin and Zinnat. In order to compare their ability to remove E. coli three paper discs, absorbed these antibiotic samples, has been put into same petri plate. Their effectiveness can be compared according to the areas of the death zones around each paper disc. From table 2, Equizolin is the most effective antibiotic against E. coli. After that, Zinnat and the least effective 8

one is Augmentin. The main cause of these differences is the different active ingredients. In Augmentin active ingredients are Amoxicillin and Clavulanic acid (URL5). On the other hand the active ingredient in Equizolin is Cefazolin Sodium (URL6). And the active ingredient of Zinnat is Cefuroxime (URL7). Because of the differences in chemical properties of these active ingredients in antibiotics, the effectiveness of them changes. The importance of antibiotics in environmental engineering processes is that if there is a mixed culture, there is a microbial competition in terms of space and nutrient. In that case, some microorganisms can produce antibiotics. In biological treatment, the kinds of microorganisms must be determined because each species have abilities to degrade different pollutants (Tortora et al.). In the third experiment, the effect of ultraviolet radiation (light) on microbial growth is measured. The UV radiation is a kind of disinfectant for microorganisms, so it is also used for removal of microorganisms (Tortora et al.). According to table 3, the effect of UV increases as the time passes because the death percentage is increasing during the time and also from the graph plotted above it can be understood because the graph is linearly increasing. The significance of UV radiation in environmental engineering processes is the same with the disinfectants. In addition to that light is also important for some photosynthetic microorganisms (Tortora et al.). 6. REFERENCES Madigan, M.T., Martinko, J.M., Stahl, D.A., Clark, D.P. Brock Biology of Microorganisms, 13th ed., Benjamin Cummings, Lake Ave., Glenview, IL, 2012, pp. 25-26, 756-794. Tortora, G.J., Funke, B.R., Case, C.L., Microbiology: An Introduction, 11th ed., Pearson Education, Inc., 2007, pp. 11-12, 182-184. URL 1: LA. EQIZOLIN (IM Flakon). http://www.1ilac.com/ilaclar/TumEkip/EQIZOLIN.htm (accessed April 2, 2013) URL 2: Augmentin Online. http://www.augmentins.com/ (accessed April 2, 2013) URL 3: Centers of Disease Control and Prevention. Factors Affecting The Efficacy Of Disinfection And Sterilization. http://www.cdc.gov/hicpac/Disinfection_Sterilization/4_0efficacyDS.html (accessed April 2, 2013) URL 4: Forensics Lab Supply. Fungi Aspergillus. http://grauhall.com/catalog/product_info.php?products_id=1063&osCsid=3811c5b d1ba1561dd0f09970bcba3947 (accessed April 2, 2013) (image source) URL 5: GLOBALRPh: The Clinicians Ultimate Reference. CEFAZOLIN (ANCEF ) Intravenous (IV) Dilution. http://www.globalrph.com/cefazolin_dilution.htm (accessed April 2, 2013) 9

URL 6: Midlands Technical College. Selected Slides for Recognition Variety of Kingdoms. http://classes.midlandstech.edu/carterp/Courses/bio225/Lab%20Stuff/skills%20slid es/1showlabslide.htm (accessed April 2, 2013) (image soruce) URL 7: Rutala, W.A. Disinfectants Used for Surface Disinfection and New Room Decontamination Technology. http://www.unc.edu/depts/spice/dis/EnvDisinfAPIC12.pdf (accessed April 2, 2013) URL 8: The Evergreen State College Forensics and Clinical Behavior. Calibration of the Ocular Micrometer on a Microscope. http://academic.evergreen.edu/curricular/fcb/wk2calibration.doc (accessed April 2, 2013) URL 9: The Ohio State University PCMB 102 Laboratory Resources. Protista > http://www.biosci.ohioGreen Algae > Cladophora. state.edu/~plantbio/osu_pcmb/pcmb_lab_resources/pcmb102_activities/algae_moss es/algae_mosses_cladophora.htm (accessed April 2, 2013) (image source) URL 10: Thompson, K. The Science Of Disinfectants. Cleaning and Maintenance Management Magazine, Feb. 8, 2012. http://www.cmmonline.com/articles/thescience-of-disinfectants-7 (Accessed April 2, 2013) URL 11: Trk la Rehberi: En Gncel la Prospektsleri. ZINNAT 500 MG 10 TABLET Prospekts Bilgileri | la Farmakolojik zellikleri. http://www.ilactr.com/ilac.php?a=zinnat_500_mg_14_tablet&i=6365 (accessed April 2, 2013) URL 12: University of Michigan Measuring Techniques III. Measuring the Size of a Specimen Using a Stage Micrometer and an Ocular Micrometer. http://slc.umd.umich.edu/slconline/MT3/MeasuringTech3.pdf (accessed April 2, 2013) URL 13: Visualphotos.com. Radiolaria, Protozoa, mineral skeletons, 200 X optical microscope, photomicrography , protozoan. http://www.visualphotos.com/image/1x7606130/radiolaria_protozoa_mineral_skele tons_200_x (accessed April 2, 2013) (image source)

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