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FACULTY OF ENGINEERINGTECHNOLOGY
Lab 05
STERILIZATION BTP 1213 BIOLOGY FOR ENGINEERS Lab Objectives By the end of this lab, students should be able to: 1. To study the effect of disinfection towards antibacterial activity. 2. To study the effect of ultraviolet ray towards bacteria 3. To study the effect of antibiotics towards bacteria
20
Due Date:
Student names
Student ID
Section
Delivery Date:
Group
BTP1213 BIOLOGY FOR ENGINEERS
2
EXPERIMENT 5: STERILIZATION 1.0 OBJECTIVES
1. To study the effect of disinfection towards antibacterial activity. 2. To study the effect of ultraviolet ray towards bacteria 3. To study the effect of antibiotics towards bacteria
2.0 INTRODUCTION
Sterilization of culture and apparatus is very important in microbiological field to avoid contamination. In other word, sterilization means disinfectant of life organisms including endospora of bacteria. All culture media are sterilized, or rendered free of all life, prior to use. In microbiology laboratory, sterilization is usually accomplished using autoclave. Containers of culture media, such as test tubes or petri plates, should not be opened until you are ready to work with them, and even then, they should not be left open.
There are several methods for sterilization namely:
a)Dry heat
Bunsen burner: Toflame the apparatus which been immersed in alcohol. (i.e. inoculating loop, slide, forceps and test tube).
Oven: Hot air oven is used to sterile glass instrument such as test tube, pipette, petri dish and flask. Sterilization will be conducted for at least 1hr to 2hours at temperature 160oC.
b)Wet heat
Boil: 5 to 10 minutes is enough to kill the microbe without spore. However, it will not killing the endospore bacteria even though its boil for several hours.
Steamer: Suitable to material that sensitive to heat over the temperature of 100oC.
Autoclave: Steamer under pressure. Autoclave is conducted at temperature of 121oC, pressure of 15ib/in2 for 15 to 30 minutes.The bottle cap should be always loose before get
BTP1213 BIOLOGY FOR ENGINEERS
3
into autoclave. The gauze wool should be closed with aluminum foil. Always tight the cap after take it out from autoclave.
Pasteurization: The heating of every particle of milk or milk product at 72oCfor 20second without allowing recontamination of that milk or milk product during the heat treatment process. This method can destroy some undesirable enzymes and many spoilage bacteria without spoiling the quality of the milk but not the endosporabacteria.This method also suitable to sterilize a glass apparatus, laboratory instrument and other contaminate material. Normally, 20 minutes is enough for sterilization process. However, if the medium used is more than 14 liter, the time should be extended.
c) Filtration
This method is for the sample solution which sensitive to heat such as vitamin, protein, enzyme, and animal cell. The pore size of the filter normally is around 0.22 µm which is suitable to remove the bacteria. However, all the apparatus should be autoclave before being used.
d) Chemical
Is normally used to disinfectant the skin surface, floor and to all apparatus that sensitive to heat.All the pipette and slide used should be place into the disinfectant medium.If the culture spill out to the floor, it should be covered with cotton that immerse in disinfectant medium before it being wash.
e) Light
Radioisotope: Is used to sterilize disposable and non-disposable apparatus. Examples for disposable apparatus arethe syringe, blade scalpel, glove, cotton, dialysis set, petri dish, and others. For non-disposable apparatus, it includes aspirin, vaccine antibiotic, eye drop acrylic powder and others.
Ultra violet: Is used to sterilize certain place such as room or laminar flow
BTP1213 BIOLOGY FOR ENGINEERS
3.0
4
EFFECT OF ANTIBACTERIAL ACTIVITY BY DISINFECTION
Materials:
Escherichia coli broth in universal bottle 2 nutrient agar plate Cotton swab Filter paper Forcep Disinfectantmedium : Dettol with dilution 2%, 1%, 0.5% and 0.25%
Methodology:
Preparation of Culture Medium
Preparation of nutrient agar: Weigh out 23g of nutrient agar powder. Add to 1L of distilled or deionized water in a 1L Schott bottle. Sterile at 121°C for 20 minutes. Cool to 50°C.
Preparation of agar plates: Pour 15-20mL of a warm sterile nutrient agar per petri plate. Allow the nutrient agar to harden.
1.
Prepare 5ml Dettol with dilution 2%, 1%, 0.5% and 0.25% respectively.
2.
Divide two sections in your nutrient agar plate and label each section with 2% Dettol and 1% Dettol. Repeat the same step by dividing another nutrient agar plate into two sections and labels those sections into 0.5% Dettol and 0.25% Dettol respectively.
3.
Spread the E. coli culture on the nutrient agar surface with aseptic techniques.
4.
By using the forcep, immerse3-4 pieces of filter paper into 2% Dettol and put it on the
surface of nutrient agar that had been labeled for 2% Dettol.
BTP1213 BIOLOGY FOR ENGINEERS
5.
Repeat the same step for 1%, 0.5% and 0.25% Dettol.
6.
Incubate the petri plates for at least 24hour at temperature of 37oC.
3.1
EFFECT OF BACTERIA TOWARDS ULTRAVIOLET RAY
5
Radiant energy comes to the Earth from the Sun and other extraterrestrial sources, and some is generated on Earth from natural and human-made source. Radiation differs in wavelength and energy. The shorter wavelengths have more energy. X rays and gamma rays are forms of ionizing radiation. Their principle effect is to ionize water into highly reactive free radicals (with unpaired electrons) that can break strands of DNA. The effect of radiation is influenced by many variables, such as the age of cells, media composition, and temperature.
Non-ionizing radiation between 15 to 390 nm is called ultraviolet (UV). Wavelengths below 200 nm are absorbed by air and do not reach living organisms. The most lethal wavelengths, sometimes called biocidal, are in the UVC range, 200 to 290 nm. These wavelengths correspond to the optimal absorption wavelengths of DNA. UVB wavelengths (290 to 320 nm) can also cause damage to DNA. UVA wavelengths (320 to 400 nm) are not as readily absorbed and are therefore less active on living organisms.
Ultraviolet light induces pyrimidine dimers in the nucleic acid, which result in a mutation. Mutations in critical genes result in the death of the cell unless the damage is repaired. When pyrimidine dimmers are exposed to visible light, photolyases are activated; these enzymes split the dimmers. This is called light repair or photoreactivation. Another repair mechanism, called dark repair, is independent of light. Dimers are removed by endonuclease, DNA polymerase replaces the bases, and DNA ligase seals the sugar-phosphate backbone.
As a sterilizing agent, ultraviolet radiation is limited by its poor penetrating ability. It is used to sterilize some heat-labile solutions, to decontaminate hospital operating rooms and foodprocessing areas, and to disinfect wastewater.As a precaution step, avoid looking directly to the UV light or sitting near the light source in order to prevent from eye damage.
BTP1213 BIOLOGY FOR ENGINEERS
6
Materials: Escherichia coli broth in universal bottle 1 sterile pipette (1 ml) Alcohol 3 nutrient agar plates
Methodology:
1.
Pipette 0.2 mlof E. coli culture in the middle of agar surface. Repeat for other 2 agar plates.
2.
Spreadthe E. coli culture with sterile glass spreader.
3.
Put all 3 plates into UV hood. For the first plate, cover the plate with an aluminium foil.For the second plate, open the lid and for the third plate, remain the plate with its lid.
4.
Switch on the UV light for 20 minute.
5.
Then, put back the lid for the second dish and remove all of them from the UV hood. Incubate all the dishes at temperature of 37˚C for 24 to 48 hours.
6.
Record the data afterone day to one week of experiment.
BTP1213 BIOLOGY FOR ENGINEERS
3.2
7
EFFECT OF BACTERIA TOWARDS ANTIBIOTICS
A variety of chemical compounds can be used to kill the microbe such as antibacterial soap, detergents, natrium lauryl sulphate, quaternary ammonium halida, phenol, HgCl and others. Basically, every chemical will show a different reaction toward the microbe.Antibiotic is one of chemotherapeutic agents, chemical which is produced synthetically or biologically that being used for curing the disease.Antibiotic is a chemical substance that produced by several microorganisms and can killonly a specific microorganism.Antibiotic is useful if:
Can be used to kill wide range of microorganisms
Have a lowest microorganisms resistance towards it
Does not give any side effect to thehost
Does not destroy the environment
There are two types of antibiotics; one that has a wide range of activity, while the other one has only a limited activity.Antibiotics that are derived by actinomycetes such as Streptomyces genus, like Penicillium produced from spore and Bacillus produced from bacteria.Antibiotics mode of reaction includes:
Inhibit the wall cell formation
Damage the membrane cell
Disturb the protein synthesis
Inhibit the nucleic acid formation
Materials: E. coli in broth Peptone yeast extract broth (24 hours) Sterile pipette (1 ml) 70 % Ethanol Nutrient agar plate Antibiotic disk Forceps
BTP1213 BIOLOGY FOR ENGINEERS
8
Methodology:
1.
Put three drops of E. coli broth to the middle of the plate using sterile Pasteur pipette. Spread it properly using a sterile glass spreader.
1.
Let the inoculums immerse into the agar about 20-30 minutes.
2.
Put the antibiotic disk on the surface of agar using sterilized forceps.
3.
Incubate the agar plate at temperature of 37˚C for 24 hours.
4.
Record the diameter of the clear area in the agar plate after one day to one week of experiment.
4.0
QUESTIONS
1.
What are the factors that affect the diameter of the inhibition zone?
2.
Why is microbial control necessary?
3.
Compare the sterilization technique and disinfection technique.
4.
How 70% ethanol and UV radiation work as antimicrobial agent?
5.
How do the antibiotics work to inhibit the growth of microorganism and control their population?
Lab 05
STERILIZATION BTP 1213 BIOLOGY FOR ENGINEERS Lab Objectives By the end of this lab, students should be able to: 1. To study the effect of disinfection towards antibacterial activity. 2. To study the effect of ultraviolet ray towards bacteria 3. To study the effect of antibiotics towards bacteria
20
Due Date:
Student names
Student ID
Section
Delivery Date:
Group
BTP1213 BIOLOGY FOR ENGINEERS
2
EXPERIMENT 5: STERILIZATION 1.0 OBJECTIVES
1. To study the effect of disinfection towards antibacterial activity. 2. To study the effect of ultraviolet ray towards bacteria 3. To study the effect of antibiotics towards bacteria
2.0 INTRODUCTION
Sterilization of culture and apparatus is very important in microbiological field to avoid contamination. In other word, sterilization means disinfectant of life organisms including endospora of bacteria. All culture media are sterilized, or rendered free of all life, prior to use. In microbiology laboratory, sterilization is usually accomplished using autoclave. Containers of culture media, such as test tubes or petri plates, should not be opened until you are ready to work with them, and even then, they should not be left open.
There are several methods for sterilization namely:
a)Dry heat
Bunsen burner: Toflame the apparatus which been immersed in alcohol. (i.e. inoculating loop, slide, forceps and test tube).
Oven: Hot air oven is used to sterile glass instrument such as test tube, pipette, petri dish and flask. Sterilization will be conducted for at least 1hr to 2hours at temperature 160oC.
b)Wet heat
Boil: 5 to 10 minutes is enough to kill the microbe without spore. However, it will not killing the endospore bacteria even though its boil for several hours.
Steamer: Suitable to material that sensitive to heat over the temperature of 100oC.
Autoclave: Steamer under pressure. Autoclave is conducted at temperature of 121oC, pressure of 15ib/in2 for 15 to 30 minutes.The bottle cap should be always loose before get
BTP1213 BIOLOGY FOR ENGINEERS
3
into autoclave. The gauze wool should be closed with aluminum foil. Always tight the cap after take it out from autoclave.
Pasteurization: The heating of every particle of milk or milk product at 72oCfor 20second without allowing recontamination of that milk or milk product during the heat treatment process. This method can destroy some undesirable enzymes and many spoilage bacteria without spoiling the quality of the milk but not the endosporabacteria.This method also suitable to sterilize a glass apparatus, laboratory instrument and other contaminate material. Normally, 20 minutes is enough for sterilization process. However, if the medium used is more than 14 liter, the time should be extended.
c) Filtration
This method is for the sample solution which sensitive to heat such as vitamin, protein, enzyme, and animal cell. The pore size of the filter normally is around 0.22 µm which is suitable to remove the bacteria. However, all the apparatus should be autoclave before being used.
d) Chemical
Is normally used to disinfectant the skin surface, floor and to all apparatus that sensitive to heat.All the pipette and slide used should be place into the disinfectant medium.If the culture spill out to the floor, it should be covered with cotton that immerse in disinfectant medium before it being wash.
e) Light
Radioisotope: Is used to sterilize disposable and non-disposable apparatus. Examples for disposable apparatus arethe syringe, blade scalpel, glove, cotton, dialysis set, petri dish, and others. For non-disposable apparatus, it includes aspirin, vaccine antibiotic, eye drop acrylic powder and others.
Ultra violet: Is used to sterilize certain place such as room or laminar flow
BTP1213 BIOLOGY FOR ENGINEERS
3.0
4
EFFECT OF ANTIBACTERIAL ACTIVITY BY DISINFECTION
Materials:
Escherichia coli broth in universal bottle 2 nutrient agar plate Cotton swab Filter paper Forcep Disinfectantmedium : Dettol with dilution 2%, 1%, 0.5% and 0.25%
Methodology:
Preparation of Culture Medium
Preparation of nutrient agar: Weigh out 23g of nutrient agar powder. Add to 1L of distilled or deionized water in a 1L Schott bottle. Sterile at 121°C for 20 minutes. Cool to 50°C.
Preparation of agar plates: Pour 15-20mL of a warm sterile nutrient agar per petri plate. Allow the nutrient agar to harden.
1.
Prepare 5ml Dettol with dilution 2%, 1%, 0.5% and 0.25% respectively.
2.
Divide two sections in your nutrient agar plate and label each section with 2% Dettol and 1% Dettol. Repeat the same step by dividing another nutrient agar plate into two sections and labels those sections into 0.5% Dettol and 0.25% Dettol respectively.
3.
Spread the E. coli culture on the nutrient agar surface with aseptic techniques.
4.
By using the forcep, immerse3-4 pieces of filter paper into 2% Dettol and put it on the
surface of nutrient agar that had been labeled for 2% Dettol.
BTP1213 BIOLOGY FOR ENGINEERS
5.
Repeat the same step for 1%, 0.5% and 0.25% Dettol.
6.
Incubate the petri plates for at least 24hour at temperature of 37oC.
3.1
EFFECT OF BACTERIA TOWARDS ULTRAVIOLET RAY
5
Radiant energy comes to the Earth from the Sun and other extraterrestrial sources, and some is generated on Earth from natural and human-made source. Radiation differs in wavelength and energy. The shorter wavelengths have more energy. X rays and gamma rays are forms of ionizing radiation. Their principle effect is to ionize water into highly reactive free radicals (with unpaired electrons) that can break strands of DNA. The effect of radiation is influenced by many variables, such as the age of cells, media composition, and temperature.
Non-ionizing radiation between 15 to 390 nm is called ultraviolet (UV). Wavelengths below 200 nm are absorbed by air and do not reach living organisms. The most lethal wavelengths, sometimes called biocidal, are in the UVC range, 200 to 290 nm. These wavelengths correspond to the optimal absorption wavelengths of DNA. UVB wavelengths (290 to 320 nm) can also cause damage to DNA. UVA wavelengths (320 to 400 nm) are not as readily absorbed and are therefore less active on living organisms.
Ultraviolet light induces pyrimidine dimers in the nucleic acid, which result in a mutation. Mutations in critical genes result in the death of the cell unless the damage is repaired. When pyrimidine dimmers are exposed to visible light, photolyases are activated; these enzymes split the dimmers. This is called light repair or photoreactivation. Another repair mechanism, called dark repair, is independent of light. Dimers are removed by endonuclease, DNA polymerase replaces the bases, and DNA ligase seals the sugar-phosphate backbone.
As a sterilizing agent, ultraviolet radiation is limited by its poor penetrating ability. It is used to sterilize some heat-labile solutions, to decontaminate hospital operating rooms and foodprocessing areas, and to disinfect wastewater.As a precaution step, avoid looking directly to the UV light or sitting near the light source in order to prevent from eye damage.
BTP1213 BIOLOGY FOR ENGINEERS
6
Materials: Escherichia coli broth in universal bottle 1 sterile pipette (1 ml) Alcohol 3 nutrient agar plates
Methodology:
1.
Pipette 0.2 mlof E. coli culture in the middle of agar surface. Repeat for other 2 agar plates.
2.
Spreadthe E. coli culture with sterile glass spreader.
3.
Put all 3 plates into UV hood. For the first plate, cover the plate with an aluminium foil.For the second plate, open the lid and for the third plate, remain the plate with its lid.
4.
Switch on the UV light for 20 minute.
5.
Then, put back the lid for the second dish and remove all of them from the UV hood. Incubate all the dishes at temperature of 37˚C for 24 to 48 hours.
6.
Record the data afterone day to one week of experiment.
BTP1213 BIOLOGY FOR ENGINEERS
3.2
7
EFFECT OF BACTERIA TOWARDS ANTIBIOTICS
A variety of chemical compounds can be used to kill the microbe such as antibacterial soap, detergents, natrium lauryl sulphate, quaternary ammonium halida, phenol, HgCl and others. Basically, every chemical will show a different reaction toward the microbe.Antibiotic is one of chemotherapeutic agents, chemical which is produced synthetically or biologically that being used for curing the disease.Antibiotic is a chemical substance that produced by several microorganisms and can killonly a specific microorganism.Antibiotic is useful if:
Can be used to kill wide range of microorganisms
Have a lowest microorganisms resistance towards it
Does not give any side effect to thehost
Does not destroy the environment
There are two types of antibiotics; one that has a wide range of activity, while the other one has only a limited activity.Antibiotics that are derived by actinomycetes such as Streptomyces genus, like Penicillium produced from spore and Bacillus produced from bacteria.Antibiotics mode of reaction includes:
Inhibit the wall cell formation
Damage the membrane cell
Disturb the protein synthesis
Inhibit the nucleic acid formation
Materials: E. coli in broth Peptone yeast extract broth (24 hours) Sterile pipette (1 ml) 70 % Ethanol Nutrient agar plate Antibiotic disk Forceps
BTP1213 BIOLOGY FOR ENGINEERS
8
Methodology:
1.
Put three drops of E. coli broth to the middle of the plate using sterile Pasteur pipette. Spread it properly using a sterile glass spreader.
1.
Let the inoculums immerse into the agar about 20-30 minutes.
2.
Put the antibiotic disk on the surface of agar using sterilized forceps.
3.
Incubate the agar plate at temperature of 37˚C for 24 hours.
4.
Record the diameter of the clear area in the agar plate after one day to one week of experiment.
4.0
QUESTIONS
1.
What are the factors that affect the diameter of the inhibition zone?
2.
Why is microbial control necessary?
3.
Compare the sterilization technique and disinfection technique.
4.
How 70% ethanol and UV radiation work as antimicrobial agent?
5.
How do the antibiotics work to inhibit the growth of microorganism and control their population?
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