LAB 3 MAYA EMIRA BT MAD SAAD 111381

PREPARATION AND STERILIZATION OF CULTURE MEDIA

INTRODUCTION

Bacteria grown in a laboratory environment, like captive animals in a zoo, need to have everything provided for them such as food, water, a suitable environment,in order to survive and thrive. Some microbes are not especially choosy in their requirements for growth, while others, such as Treponema pallidum, the causative agent of syphilis, has never been successfully grown in culture, although scientists have been trying to do so for more than 100 years.

Several basic types of media are discussed below. Although their differences are featured, there are several characteristics that all culture media have in common. Media must be prepared in such a way that it is sterile prior to being inoculated with a bacterial sample, so that when a particular type of bacteria is cultured (cultivated) on that medium, it is the only type of bacteria present. Growth media must also provide everything the bacterial culture needs to live and grow, including water, nutrients, and the proper pH. Media can be eitherliquid (nutrient broth) or solid (agar).

Most clinical cultures do not have such exacting requirements, and can be grown in what is referred to as “complex media”. Complex media are composed of partially digested yeast, beef, soy and additional proteins, in which the exact concentration and composition is unknown. In comparison with defined media, which are good for growing bacteria with very particular needs, complex media can be thought of as a crowd-pleaser, suitable for growing many different types of less fastidious microbes.

In addition to growth media formulations being classified as either defined or complex, there are also media that are designed to do more than just grow bacteria, selective and differential media provide information about the bacteria growing.

Selective media contain ingredients that inhibit the growth of certain types of bacteria and encourage the growth of others. This type of media is useful in helping to identify unknown bacteria and in encouraging the growth of only the types of bacteria that the microbiologist is interested in cultivating.

For example MacConkey’s Agar (MAC) is used to cultivate Gram-negative bacteria, by discouraging the growth of Gram positive bacteria through the use of crystal violet dyes and bile salts. Mannitol Salt Agar (MSA) has a high concentration of sodium chloride, which selects for halophiles (salt-loving bacteria) such as members of the genus Staphylococcus.

Differential culture media are formulated to display a color change when the bacteria growing utilize a certain ingredient. For example MacConkey's Agar, in addition to being selective, contains the sugar lactose and a pH sensitive dye. When bacteria growing on MAC ferment lactose (metabolize it for food), they generate waste products that trigger the pH sensitive dye to turn the bacteria pink.
Mannitol Salt Agar also contain food (mannitol, a sugar alcohol) and a pH sensitive dye. When the bacteria growing on MSA ferment mannitol, the medium changes from its original pink color to a bright yellow. Another specialized medium, Blood Agar (BAP) contains sheep’s blood, if bacteria growing on the medium produce exotoxins that hemolyze (cut up blood cells), the medium changes color.

Culture media must be stored at the specified temperature, under specified conditions and not longer than the shelf-life periods appropriate to each product. The storage conditions and expiry date of each product are shown on the labels or product inserts but the following general rules will help to ensure that they are kept in an optimum environment. When storing products note the shelf life expiry dates on the labels and use the products in order of their lot/batch numbers.

Light

All prepared culture media and their components should be stored away from light and exposure to direct sunlight should be avoided at all times.

Humidity

Sealed glass and plastic containers are unaffected by normal laboratory humidity. Opened containers of dehydrated powders will be affected by high humidity. Hot, steamy media preparation rooms are not suitable environments to store containers of culture media; particularly containers which are frequently opened and closed. An adjacent cold room or an adequate storage cupboard are preferable storage areas.

Temperature and time

Culture Media:  Sealed, unopened containers should be stored at room temperature 15-20°C. Opened containers should have the cap or lid carefully and securely replaced. It is important that opened containers are stored in a dry atmosphere at room temperature. Shelf life 1 to 5 years..

 

OBJECTIVES

To prepare sterile nutrient agar for culturing microorganism

DISCUSSION

An autoclave is an instrument used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents.

Autoclaves are widely used in microbiology,medicine,mycology,dentistry,chiropody and prosthetics fabrication. 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 hospital 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, etc. 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.


Autoclaves are also widely used to cure composites and in the vulcanization of rubber. The high heat and pressure that autoclaves allow help to ensure that the best possible physical properties are repeatably attainable.
It is very important to ensure that all of the trapped air is removed from the autoclave before activation, as hot air is a very poor medium for achieving sterility. Steam at 134 °C can achieve in three minutes the same sterility that hot air at 160 °C takes two hours to achieve. Methods of achieving air removal include:

 Downward displacement (or gravity-type) - As steam enters the chamber, it fills the upper areas first as it is less dense than air. This compresses the air to the bottom, forcing it out through a drain which often contains a temperature-sensing device. Only when air evacuation is complete does the discharge stop. Flow is usually controlled by a steam trap or a solenoid valve, but bleed holes are sometimes used, often in conjunction with a solenoid valve. As the steam and air mix it is also possible to force out the mixture from locations in the chamber other than the bottom.

Steam pulsing - air dilution by using a series of steam pulses, in which the chamber is alternately pressurized and then depressurized to near atmospheric pressure.

Vacuum pumps - a vacuum pump sucks air or air/steam mixtures from the chamber.

Superatmospheric cycles - achieved with a vacuum pump. It starts with a vacuum followed by a steam pulse followed by a vacuum followed by a steam pulse. The number of pulses depends on the particular autoclave and cycle chosen.

Subatmospheric cycles - similar to the superatmospheric cycles, but chamber pressure never exceeds atmospheric pressure until they pressurize up to the sterilizing temperature.

 

REFERENCES

Bauman, R. (2007). Microbiology with Diseases by Taxonomy. Pearson Benjamin Cummings.

Perry, J. and Stanley, J. (1997) Microbiology Dynamics & Diversity. Saunders College Publishing.

http://www.protocol-online.org/biology-forums-2/posts/18634.html

http://www.cabri.org