Lab 3 report written by Yu Tiam Meng

Lab 3 report written by Yu Tiam Meng

Name: Yu Tiam Meng

Matric card no.: 111437

Lab 3: Preparation and sterilization of culture media

Introduction:

Culture media are available commercially as powders; they require only the addition of water. Nutrient medium is a general purpose preparation for culturing microorganisms which are not nutritionally fastidious. The broth contains:

3.0g/L “Lab – lemco” power ( a beef extract )

2.0g/L yeast extract

2.0g/L peptone( a nitrogen source)

5.0g/L sodium chloride

15.0g/L agar powder

  

  The ready-made nutrient agar contains 15 g/L agar and the same contents as the manually-made nutrient medium. The final pH of both media has to be 7.4.

    

  Autoclaving is a process that use moist heat and pressure so thst all parts of the material to be sterilized reach 121 ̊C for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurixed steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented.

  

   Steam is continually forced into the chamber until the pressure reaches 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121 ̊C. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 ̊C throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilixed objects can then be removed.

Objective:

To prepare sterile nutrient agar for culturing microorganisms

Material and reagents:

Ready-made commercial nutrient agar
Balance
Distilled water
Scott bottles
Measuring cylinder
Beaker
Universal bottles

Discussions:

1. Steps of carry out autoclaving(sterilization of culture media):

1. Preparing the Items.
   • Prepare all items to be sterilized. Pyrex glassware is the only type of glassware that should be placed in an autoclave. Liquids should be placed in approved glassware with lids on but loosened to allow pressure to be released. Tin foil can also be used to loosely cover glassware openings. Only liquids such as cell culture media and water should be placed in an autoclave.
     Tools such as tweezers, scalpels and other small lab equipment should be placed into approved autoclave plastic bags. Any solid materials should be put into an approved container before being placed into the plastic bags.
     Place all bagged items and glassware into an approved autoclave plastic or metal tub. If using a plastic tub, fill the tub with 1 to 2 inches of distilled water. Do not stack bags or items on top of one another in the tub.
   Starting the Autoclave
   • Open the autoclave door slowly. Wait at least 15 minutes after the last autoclave run before opening the door, as pressure and heat can still be captured inside. Slowly open the door while firmly holding the door handle.
     Fill the bottom of the autoclave compartment with distilled water up to the fill line. Double-check all plastic bags and glassware to make sure they are placed flat in the tub.
     Close the door and engage the lock. Make sure the power switch is set to "on" and the steam supply valve is open. Set the timer to the desired time. Fifteen to 20 minutes is usually sufficient to sterilize non-filled glassware and metal lab supplies. Thirty minutes is good for filled glassware and 60 minutes is required for biohazard waste.
     Set the cycle to "Unwrapped," "Wrapped" or "Liquids." The autoclave in your lab may vary in setting types; consult with the lab staff if your machine is labeled differently. "Unwrapped" is for items that aren't in plastic bags.
     Double-check the door is shut and locked before pressing "start." Wait until the PSI gauge is at 15 before leaving the room.
   Removing Items
   • When the cycle is over, wait at least 15 minutes before opening the door. Have a stainless steel lab rack nearby to place the tubs on, as they will be very hot. Put on protective eyewear, lab smock, and heavy duty heat and flame resistant lab gloves.
     Wait until the PSI gauge is at 0 before you slowly release the lock and open the door. Carefully reach inside and firmly grasp the tub on each side. Slowly slide it out, keeping your face away from the tub. Place it on the rack and wait for the items to cool down.

2. Principle of Laminar Air Flow

Laminar Air Flow is based on the flow of air current to create uniform velocity, along parallel lines, which helps in transforming microbial culture in aseptic conditions .

Conclusion: 

I have learned how to prepare the culture medium with all the ingredients listed and also precautions that needed to be take into account. I have also learned the sterilization of culture medium by autoclaving and some important precautions about it.

References:

http://www.ehow.com/way_5589124_autoclave-sterilization-procedures.html 

http://wiki.answers.com/Q/What_is_the_principle_of_Laminar_air_flow

http://www.globalrph.com/aseptic.htm

LAB REPORT 3
PREPARED BY ISKANDAR.
Name:syed iskandar 
Metric no:111434

Lab 3: Preparation and sterilization of culture media

Introduction:

Culture media are available commercially as powders; they require only the addition of water. Nutrient medium is a general purpose preparation for culturing microorganisms which are not nutritionally fastidious. The broth contains:

3.0g/L “Lab – lemco” power ( a beef extract )   

2.0g/L yeast extract      

2.0g/L peptone( a nitrogen source)

5.0g/L sodium chloride

15.0g/L agar powder

  

  The ready-made nutrient agar contains 15 g/L agar and the same contents as the manually-made nutrient medium. The final pH of both media has to be 7.4.

    

  Autoclaving is a process that use moist heat and pressure so thst all parts of the material to be sterilized reach 121 ̊C for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurixed steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented.

  

   Steam is continually forced into the chamber until the pressure reaches 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121 ̊C. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 ̊C throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilixed objects can then be removed.

Objective:

To prepare sterile nutrient agar for culturing microorganisms

Material and reagents:

Ready-made commercial nutrient agar
Balance
Distilled water
Scott bottles
Measuring cylinder
Beaker
Universal bottles

Discussion:

autoclaving procedure:

• Fill liquid containers only half full.
• Loosen caps or use vented closures.
• Always put bags of biological waste into pans to catch spills.
• Position biohazard bags on their sides, with the bag neck taped loosely.
• Leave space between items to allow steam circulation.
• Household dishpans melt in the autoclave. Use autoclavable polypropylene or stainless steel pans.

laminar flows:

Laminar air flows can maintain a working area devoid of contaminants. Uses Laminar Flow Cabinets are suitable for a variety of applications and especially where an individual clean air environment is required for smaller items, e.g. particle sensitive electronic devices. In the laboratory, Laminar Flow Cabinets are commonly used for specialised work. Laminar Flow Cabinets can be tailor made to the specific requirements of the laboratory and are also ideal for general lab work, especially in the medical, pharmaceutical, electronic and industrial sectors. How They Work The process of laminar air flow can be described as airflow where an entire body of air flows with steady, uniform velocity. Laminar Flow Cabinets work by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection. Air is taken through a filtration system and then exhausted across the work surface as part of the laminar flows process. Commonly, the filtration system comprises of a pre-filter and a HEPA filter. The Laminar Flow Cabinet is enclosed on the sides and constant positive air pressure is maintained to prevent the intrusion of contaminated room air. Horizontal Laminar Flow Cabinets Horizontal Laminar Flow Cabinets receive their name due to the direction of air flow which comes from above but then changes direction and is processed across the work in a horizontal direction. The constant flow of filtered air provides material and product protection. Vertical Laminar Flow Cabinets Vertical Laminar Flow Cabinets function equally well as horizontal Laminar Flow Cabinets with the laminar air directed vertically downwards onto the working area. The air can leave the working area via holes in the base. Vertical flow cabinets can provide greater operator protection. They come with either horizontal or vertical filtered laminar air flow. The horizontal clean bench pushes air through a HEPA filter and directs the filtered air horizontally over the work surface at a constant speed, toward the operator. A vertical clean bench pushes air through a HEPA filter which is located at the top of enclosure. The air is directed downward onto the work surface at a constant speed. Both configurations offer product protection…so why choose one over the other? Application. Horizontal clean benches generally have a taller and therefore a larger loading and working area. This allows for instruments, like microscopes to be placed inside. They are typically the preferred selection for plant tissue culture, media plate preparation, electronics inspection, medical device assembly and pharmacy drug preparation. Because they do not provide protection to the user, they should not be used in conjunction with biohazardous material, toxins or radionuclides. One of the key benefits is the absence of a sash, which is an advantage when placing instruments and equipment inside. A vertical clean bench, on the other hand, offers a pivoting glass sash as a safety measure. This sash offers personal protection and a physical barrier when working with chemicals. Vertical clean benches are common place in microbiology, forensic, biotech and many other industries, which utilizes them during procedures that require clean work area.

conclusion:

i have learned the procedure on using the autoclave and how to use a laminar flow.I also learned on the precautions on using the autoclave and laminar flow.








LAB 3 WRITTEN BY AZIZUL


Name : Ahmad Azizul Bin Md Sadik
Matric No : 114116

LAB 3

PREPARATION AND STERILIZATION OF CULTURE MEDIA

Introduction

Culture media are available commercially as powders. It only require the addition of water. Nutrient medium is general purpose preparation for culturing microorganisms which are not nutritionally fastidious. The broth contain :

• 3.0 g/L “Lab-lemco” powder (beef extract)
• 2.0 g/L yeast extract
• 5.0 g/L peptone (nitrogen source)
• 5.0 g/L sodium chloride
• 2.0 g/L agar powder

The agar has the same composition, except that it contains 15 g/L agar. The final pH of both media is 7.4.

Moist heat in the form of pressurized steam is regarded as the most dependable method for the destruction of all forms of life, including bacterial spores. Autoclave is one of the processes that use moist heat and pressure on all parts of the materials to be sterilized. Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121^oC) will kill all organisms and their endospores in about 15 minutes.

A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. For example, when free flowing steam at a temperature of 100^oC is placed under a pressure of 1 atmosphere above sea level pressure, that is, about 15 pounds of pressure per square inch (Psi), the temperature rises to 121^oC. Increasing the pressure to 20 psi raises the temperature to 126^oC. In this way steam is a gas, increasing its pressure in a closed system increases its temperature. As the water molecules in steam become more energized, their penetration increases substantially. This principle is used to reduce cooking time in the home pressure cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing agent is the moist heat, not the pressure.

Objective :

To prepare sterile nutrient agar for culturing miroorganisms.

Materials and Reagents :

• Commercial nutrient agar
• Balance
• Distilled water
• Scott bottles

Discussion :

Autoclaves are widely used in microbiology, medicine, tattooing, body piercing, veterinary science, mycology, dentistry, chiropody and prostheticsfabrication. They vary in size and function depending on the media to be sterilized. Typical loads include laboratory glassware, surgical instruments, medical waste, patient pair utensils, animal cage bedding, and lysogeny broth. A notable growing application of autoclaves is the pre-disposal treatment and sterilization of waste material, such as pathogenic waste. Machines in this category largely operate under the same principles as conventional autoclaves in that they are able to neutralize potentially infectious agents by utilizing pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressing, gloves. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven. For all-glass syringes, sterilizing in a hot air oven is a better method.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions. For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. Alternatively steam penetration can be achieved by shredding the waste in some Autoclave models that also render the end product unrecognizable. During the initial heating of the chamber, residual air must be removed. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there. To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of time.

The original parent company, the Liebig Extract of Meat Company (Lemco) was formed in 1865 and manufactured meat infusion extracts under the trade name Lab Lemco. This could conveniently be used in laboratories to grow bacteria. Early biologists’ generally attempted to grow microorganisms using the food or sample on which the organism had first been observed, such as Bartolomeo Bizio’s 1832 study of ‘blood spots’ on communion wafers, caused by Serratia marcescens, which used bread as a growth medium. However, when dealing with non-pigmented organisms and pathogens, Robert Koch found that broths based on fresh beef serum or meat extracts (bouillions) gave the best growth. Although meat extract is a valuable source of many growth factors for bacteria it lacks sufficient amino nitrogen to allow optimal growth of a range microorganisms. In 1884 Fredrick Loeffler added peptone and salt to Koch’s basic meat extract formulation. The peptone he used was an enzymatic digest of meat, produced in the 19th Century as a pharmaceutical product, usually prescribed for nutritional disorders. This peptone added amino-nitrogen, while the salt raised the osmolarity of the medium.

Yeast Extract is an autolysate of yeast cells used in preparing microbiological culture media. Yeast Extract is the water-soluble portion of autolyzed yeast. The autolysis is carefully controlled to preserve naturally occurring B-complex vitamins. Yeast Extract is prepared and standardized for bacteriological use and cell cultures, and is an excellent stimulator of bacterial growth. Yeast Extract is generally employed in the concentration of 0.3% - 0.5%. Yeast Extract is typically prepared by growing baker’s yeast, Saccharomyces spp., in a carbohydrate-rich plant medium. The yeast is harvested, washed, and resuspended in water, where it undergoes autolysis. Yeast Extract is the total soluble portion of this autolytic action. The autolytic activity is stopped by a heating step. The resulting Yeast Extract is filtered clear and dried into a powder by spray drying. Yeast Extract has been successful in culture media for bacterial studies in milk and other dairy products. Several media containing Yeast Extract have been recommended for cell culture application.

Bacteria require nitrogen to make proteins and nucleic acids. Only a few genera of bacteria can use free molecular nitrogen from the air. Others require fixed nitrogen in the organic or inorganic form. Some can use nitrate or nitrite salts, but most require amino acids, peptides, peptones, or proteins. Peptones are the most widely used source of nitrogen in microbial media. Peptones are enzymic digests of other proteins often meat scraps. Some are made by cooking milk or meat products in acid, but most are made by incubating milk or meat with trypsin, pepsin, or other proteolytic enzymes to digest the protein to a mixture of amino acids, peptides, and polypeptides. Many microbes, called proteolytic, can digest proteins, but most can't. The choice of peptone is sometimes of importance. Tryptones are the best choice for bacteria media because they are used by most bacteria from animals and supply nitrogen, energy, and carbon. Tryptone water (tryptone + water) will support the growth of many species of bacteria. Tryptones are not pure substances. They are a mixture of left over trypsin, salts, and vitamins, amino acids, peptides, peptones (longer than peptides), and polypeptides (longer than peptones). Tryptones are used in foods for flavoring and nutrition, in electroplating for smooth plating, and in media for microbes.

Agar is used throughout the world to provide a solid surface containing medium for the growth of bacteria and fungi. Microbial growth does not destroy the gel structure because most microorganisms are unable to digest agar. Agar is typically sold commercially as a powder that can be mixed with water and prepared similarly to gelatin before use as a growth medium. Other ingredients are added to the agar to meet the nutritional needs of the microbes. Many specific formulations are available, because some microbes prefer certain environmental conditions over others. Agar is purified from red algae in which it is an accessory polysaccharide (polygalacturonic acid) of their cell walls.  Agar is added to microbiological media only as a solidification agent.  Agar for most purposes has no nutrient value.  Agar is an excellent solidification agent because it dissolves at near boiling but solidifies at 45^oC.  Thus, one can prepare molten (liquid) agar at 45^oC, mix cells with it, then allow it to solidify thereby trapping living cells.  Below 45^oC agar is a solid and remains so as the temperature is raised melting only when >95^oC is obtained.

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive device. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The cabinet is usually made of stainless steel with no gaps or joints where spores might collect. Laminar air flows can maintain a working area devoid of contaminants. Uses Laminar Flow Cabinets are suitable for a variety of applications and especially where an individual clean air environment is required for smaller items. In the laboratory, Laminar Flow Cabinets are commonly used for specialised work. Laminar Flow Cabinets can be tailor made to the specific requirements of the laboratory and are also ideal for general lab work, especially in the medical, pharmaceutical, electronic and industrial sectors.

The process of laminar air flow can be described as airflow where an entire body of air flows with steady, uniform velocity. Laminar Flow Cabinets work by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection. Air is taken through a filtration system and then exhausted across the work surface as part of the laminar flows process. Commonly, the filtration system comprises of a pre-filter and a HEPA filter. The Laminar Flow Cabinet is enclosed on the sides and constant positive air pressure is maintained to prevent the intrusion of contaminated room air. Horizontal Laminar Flow Cabinets Horizontal Laminar Flow Cabinets receive their name due to the direction of air flow which comes from above but then changes direction and is processed across the work in a horizontal direction. The constant flow of filtered air provides material and product protection. Vertical Laminar Flow Cabinets Vertical Laminar Flow Cabinets function equally well as horizontal Laminar Flow Cabinets with the laminar air directed vertically downwards onto the working area. The air can leave the working area via holes in the base. Vertical flow cabinets can provide greater operator protection. They come with either horizontal or vertical filtered laminar air flow. The horizontal clean bench pushes air through a HEPA filter and directs the filtered air horizontally over the work surface at a constant speed, toward the operator. A vertical clean bench pushes air through a HEPA filter which is located at the top of enclosure. The air is directed downward onto the work surface at a constant speed.

Horizontal clean benches generally have a taller and therefore a larger loading and working area. This allows for instruments, like microscopes to be placed inside. They are typically the preferred selection for plant tissue culture, media plate preparation, electronics inspection, medical device assembly and pharmacy drug preparation. Because they do not provide protection to the user, they should not be used in conjunction with biohazardous material, toxins or radionuclides. One of the key benefits is the absence of a sash, which is an advantage when placing instruments and equipment inside. A vertical clean bench, on the other hand, offers a pivoting glass sash as a safety measure. This sash offers personal protection and a physical barrier when working with chemicals. Vertical clean benches are common place in microbiology, forensic, biotech and many other industries, which utilizes them during procedures that require clean work area.

Conclusion :

As the conclusion, the objective of this experiment was achieved, that is preparing sterile nutrient agar for culturing miroorganisms. The culture media that was used for preparing the agar was the mixture of “Lab-lemco” powder (beef extract), yeast extract, peptone (nitrogen source), sodium chloride and agar powder. A few step of autoclaving also been done for sterilization of the media. Finally, the principle of laminar air flow was been explained after the process of autoclaving.

References :

• http://www.labnews.co.uk/features/history-of-the-agar-plate/
• http://en.wikipedia.org/wiki/Autoclave
• http://www.neogen.com/Acumedia/pdf/ProdInfo/7184_PI.pdf
• http://www.disknet.com/indiana_biolab/b041.htm (Revision #2 - 1999 January 2. Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164)
• http://en.wikipedia.org/wiki/Laminar_flow_cabinet
• http://www2.fiu.edu/~makemson/MCB2000Lab/Exp3MediaPrep.pdf
• http://en.wikipedia.org/wiki/Agar
• http://microbiologyon-line.blogspot.com/2009/08/autoclaving-real-sterilization.html

Lab 3 Report - by Muhammad Aizat

Name: Muhammad Aizat

Matric card no.: 111385

Lab 3: Preparation and sterilization of culture media

Introduction:

Culture media are available commercially as powders; they require only the addition of water. Nutrient medium is a general purpose preparation for culturing microorganisms which are not nutritionally fastidious. The broth contains:

3.0g/L “Lab – lemco” power ( a beef extract )

2.0g/L yeast extract

2.0g/L peptone( a nitrogen source)

5.0g/L sodium chloride

15.0g/L agar powder

 

  The ready-made nutrient agar contains 15 g/L agar and the same contents as the manually-made nutrient medium. The final pH of both media has to be 7.4.

   

  Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121 ̊C for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurixed steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented.

 

   Steam is continually forced into the chamber until the pressure reaches 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121 ̊C. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 ̊C throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilixed objects can then be removed.

Objective:

To prepare sterile nutrient agar for culturing microorganisms.

Discussions:

Microrganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have adapted to the habitats most suitable for their needs, in the laboratory, however, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. Depending on the type and combination of nutrients, different categories of media can be made.

Categories

Complex media are rich in nutrients, they contain water soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone). Usually a sugar, often glucose is added to serve as the main carbon and energy source. The combination of extracts and sugar creates a medium which is rich in minerals and organic nutrients, but since the exact composition is unknown, the medium is called complex.

Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known. Typically, they contain a simple sugar as the carbon and energy source, an inorganic nitrogen source, various mineral salts and if necessary growth factors (purified amino acids, vitamins, purines and pyrimidines).

Selective/differential media are media based on either of the two categories above supplemented with growth-promoting or growth-inhibiting additives. The additives may be species- or organism-selective (e.g., a specific substrate, or an inhibitor such as cyclohexamide (artidione) which inhibits all eucaryotic growth and is typically used to prevent fungal growth in mixed cultures).

Media	Purpose
Complex	Grow most heterotrophic organisms
Defined	Grow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays
Selective	Suppress unwanted microbes, or encourage desired microbes
Differential	Distinguish colonies of specific microbes from others
Enrichment	Similar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population
Reducing	Growth of obligate anaerobes

The mixture of necessary nutrients can be used as a liquid medium, or a solidifying agent can be added. "Agar agar" is a natural polysaccharide produced by marine algae and is the most commonly used solidifying agent added to media (end concentration usually 1.5 % w/v). If hydrolysis of the agar is suspected, a silica gel is used as a replacement solidifying agent.

Preparation of culture media

Powdered media are extremely hygroscopic and must be protected from atmospheric moisture. If possible the entire contents of each package should be used immediately after opening. Preparing the medium in a concentrated form is not recommended as some salt complexes may precipitate. Supplements that are added to the medium may affect shelf life and storage conditions. The steps for preparing the culture medium are:

1          Prepare the medium in a vessel  like beaker  that are about twice the final volume of the medium to allow adequate mixing. Weigh amount of broth and agar powder needed  into a vessel.

2          Dissolve with distilled water. Always use freshly prepared distilled or deionised water. Use warm (50°C) water to hasten the solution of the medium. Rinse all glassware with the distilled/deionised water and make sure that the vessels are clean and free from toxic chemicals which may be absorbed on to the surface of the glass e.g. bile salts, tellurite, selenite etc.

3          Heat gently, and stir until the mixture were dissolved well. Media containing agar should be heated before autoclaving. After cooldown pour the mixture into Scott bottles and loosely recap the bottles and st aside for sterilization.

Sterilization of culture media

Sterilization of culture media is best carried out in a steam autoclave at temperatures 121°C  for 15 minutes , temperature above than that may caused damage to the medium by the heating process. Heat-treatment of complex culture media which contain peptides, sugars, minerals and metals results in nutrient destruction, either by direct thermal degradation or by reaction between the medium components. Toxic products caused by chemooxidation can also be formed during heat-treatment. It is important, therefore, to optimise the heating process so that a medium is sterile after heating but minimal damage is caused to the ingredients of the medium. Autoclaves vary in performance, however, and thermocouple tests using different volumes of media should be carried out to determine the 'heat-up and 'cool-down' times.

It will be essential to do this when volumes of media greater than two litres are prepared. In order to avoid overheating large volume units of media, the 'heat-up’ and 'cool-down' periods are normally integrated into the 121°C holding time.

Sterilization Cycle

The sterilization cycle can be divided into its four stages:

·         Stage 1: 20°-121°C Chamber heat-up time

The chamber heat-up time depends on the efficiency of the autoclave (air discharge/steam input) and the size of the load in the chamber. The time required for this stage is measured with a recording probe located in the air-discharge valve located in the base of the chamber.

·         Stage 2 :<100°-121°C Heat penetration time of the medium container

The heat penetration time depends mainly on the volume of the individual containers, although the shape and the heat-transfer properties of the containers may affect this stage. The time required for the medium volume to reach 121°C is measured with thermocouples placed in the centre of the innermost container. These times assume that agar media have been dissolved before autoclaving. It is also assumed that maximum exposure to steam is possible. Thus although the single l00 ml bottle required 12 minutes to reach 121°C, when placed in a crate with other bottles it required 19 minutes and when placed in the centre of stacked crates it required 30 minutes.

·         Stage 3: 121°-121°C Holding time at the prescribed temperature

The holding time at 121°C depends on (i) the number of organisms originally present in the medium (ii) the fractional number of an organism presumed present after heating e.g. N = 0.001 equivalent to one bottle in every 1000 bottles heated becoming contaminated (iii) the thermal death rate constant of the presumed organism present at 121°C.

·         Stage 4: 121°- 80°C Cool-down time for the chamber to reach 80°C 

The cool-down time depends on the size of the load in the chamber and the heat loss rate from the autoclave. Water-sprays are used to accelerate cooling in commercial sterilizers but very careful control is required to avoid bottle fracture and the ingress of the cooling spray into the sterilized medium. The latter problem occurs when the vacuum formed in the head-space during cooling sucks contaminated cooling fluid up the thread of the cap and into the bottle. Culture media autoclaves should be unlagged and of moderate chamber capacity only. Thermal locks on the doors should prevent them opening when the chamber temperature is above 8O°C but even in these circumstances care should be taken to avoid sudden thermal shock when removing glass bottles of hot liquid from the autoclave. When screw-capped containers are placed in an autoclave the caps should be a half-turn free to allow the escape of heated air. When removed from the autoclave the containers should be allowed to cool down in a laminar airflow cabinet. Alternatively screw-capped containers may be sterilized in a jar which is covered by a piece of felt which effectively protects the containers from infection by air-borne microorganisms. Caps are screwed down tightly after the contents have cooled to ambient temperature.

Sterilization checks

All autoclaves should be checked at fixed periods of time to ensure that they are functioning efficiently. Physical measurements should be made on temperature and pressure readings, the quality of the steam should be checked, the efficiency of the 'near-to-steam' air traps in the base of the autoclave should be determined and the safety valves checked. Mandatory inspections of autoclaves as pressure vessels are normally carried out annually by specialists under instructions from insurers of such apparatus. With small laboratory autoclaves this inspection is not mandatory.

Chemical indicators will show the temperature reached or exceeded and some will indicate the time held at the specified temperature. Under-autoclaving is usually self-evident because failure to destroy all the bacterial spores naturally present in dehydrated media (the 'bioburden') will allow growth to take place in the stored or incubated medium. Failure of sterilization should always be suspected when contamination of prepared media occurs with sporing organisms. Biological indicators of sterilization will demonstrate the ability of the autoclave to destroy bacterial spores.

Autoclaving Procedures

Preparation and Loading of Materials

·         Fill liquid containers only half full.

·         Loosen caps or use vented closures.

·         Always put bags of biological waste into pans to catch spills.

·         Position biohazard bags on their sides, with the bag neck taped loosely.

·         Leave space between items to allow steam circulation.

·         Household dishpans melt in the autoclave. Use autoclavable polypropylene or stainless steel pans.

Cycle Selection

·         Use liquid cycle (slow exhaust) when autoclaving liquids, to prevent contents from boiling over.

·         Select fast exhaust cycle for glassware.

·         Use fast exhaust and dry cycle for wrapped items.

Time Selection

·         Take into account the size of the articles to be autoclaved. A 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 mL flasks each containing 250 mL of liquid.

·         Material with a high insulating capacity (animal bedding, high sided polypropylene containers) increases the time needed for the load to reach sterilizing temperatures.

·         Autoclave bags containing biological waste should be autoclaved for 50 minutes to assure decontamination.

Removing the Load

·         Check that the chamber pressure is zero.

·         Wear lab coat, eye protection, heat insulating gloves, and closed-toe shoes.

·         Stand behind door when opening it.

·         Slowly open door only a crack. Beware of rush of steam.

·         After the slow exhaust cycle, open autoclave door and allow liquids to cool for 20 minutes before removing.

Storage of prepared media

·         The recommended shelf-life of prepared culture media varies considerably. Screw-capped bottles of nutrient broth and agar can be stored for 6 months at low ambient temperatures (12-l6°C). It is important to store all media away from light. Agar plates should be stored at 2-8°C in sealed containers to avoid loss of moisture. Do not freeze.

·         Fresh media are better than stored media therefore avoid long storage times. Some very labile beta-lactam selective agents have very short active lives and media containing such substances should be used within a few days of preparation.

·         It is good laboratory practice to establish shelf-lives for all prepared media and date-stamp the containers or holders accordingly.

·         Loss of moisture from agar plates is a common cause of poor bacteriological performance. Do not preincubate all plates overnight as a sterility check. Only obviously wet plates require pre-inoculation drying.

·         Ensure that all plates are incubated in a humid environment.

·         Examine prepared media before inoculation. Look for evidence of contamination, uneven filling or bubbles on surface of agar, colour changes, haemolysis and signs of dehydration such as shrinking, cracking and loss of volume. Discard any defective plates or tubes.

Conclusion

As a conclusion ,the correct methods to prepare and sterile nutrient agar for culturing microorganisms were identified. The sterile media were prepared with taking consideration of some precautions. While the storage of prepared media, the light, humidity, temperature and time need to be consider.

References

·         http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html

·         http://www.sigmaaldrich.com/analytical-chromatography/microbiology/learning-center/theory/media-preparation.html

·         http://www.microbiol.unimelb.edu.au/staff/facilities/autoclave.html#safe