metu enve202 group 1.6 report of exp. 5

7/30/2019 METU ENVE202 Group 1.6 Report of Exp. 5

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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


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1. PURPOSEThe 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 andE.coli culture is spread on their surfacesin order to get a dense culture.

Four test tubes, containing 9 mL sterile water, are taken and 1 mL ofconcentrated 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 (1mLzefirol+9mL water).

Four paper disks are taken, soaked in each dilution tube and placed in themiddle 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 theday.

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 thenext 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 thenext day.

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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 micrometerequals 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 likewater 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)

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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 substancesName of antibiotic Spectrum Effective substance

Augmentin Bacillus anthracis,

Corynebacterium species,

Enterococcus faecalis,

Listeria monocytogenes,

Nocardia asteroides,

Streptococcus, Clostridium,

Peptococcus, Bordetella

pertussis, Brucella species,

Gardnerella

vaginalis, Legionellaspecies, Neisseria,

Pasteurella, Proteus,

Salmonella, Shigella, Vibrio

cholerae, Yersinia

enterocolitica, Bacteroides

species, Fusobacterium,

Borrelia burgdorferi,

Chlamydiae, Leptospira

icterohaemorrhagiae,

Treponema pallidum (URL

2)

Amoxicillin and Clavulanic

acid (URL 2)

Equizolin Staphylococcus aureus Cefazolin sodium (URL 3)

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(including -lactamase-

producing strains),

Staphylococcus epidermidis,

Streptococcus pyogenes,

Streptococcus agalactiae,and other strains of

streptococci, Streptococcus

pneumonia,Escherichia

coli, Proteus mirabilis (URL

1)

Zinnat Haemophilus influenzea,

Haemophilus

parainfluenzae, Moraxella

(Branhamella) catarrhalis,

Neisseria gonorrhoeae,

Escherichia coli, Klebsiellastrains, Proteus mirabilis,

Providencia strains, Proteus

rettgeri, Staphylococcus

aureus, Staphylococcus

epidermidis, Streptococcus

pyogenes, Streptococcus

pneumoniae, Streptococcus

agalactiae, Borrelia

burgdorferi,

Propionibacterium strains,

gram-negative bacilli, gram-

positivi bacilli, gram-

positive and gram-negative

cocci (URL 11)

Cefuroxime axetil (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

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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 & CALCULATIONSEffect of Disinfectant on Bacterial Growth

Table-2: Effect of disinfectant Zefirol at different concentration values

Dilution Ratio Death Rate

10-1 +++

10-2 ++

10-3 +

10-4 -

Effect of Antibiotics on Bacterial Growth

Table-3: Antibiotics death rate

Antibiotic Death Rate

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 percentageTime (min) Death (%)

0 0

5 20

15 50

20 65

30 95

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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

0; 0

5; 20

15; 50

20; 65

30; 95

y = 3.1228x

R = 0.998

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35

DeathPercentage(%)

Exposure Time (min)

Death percentage (%) vs Exposure time (min)

death

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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)

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

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

5. DISCUSSION & CONCLUSIONIn 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 forisolate 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

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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 formicroorganisms, 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 ofMicroorganisms, 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.

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metu enve202 group 1.6 report of exp. 5

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