Introduction to Lab 6: Ex. Preparation of Culture Media Media facts Preparation Sterilization Aseptic tech

1. Introduction to Lab 6: Ex. Preparation of Culture Media Media facts Preparation Sterilization Aseptic technique

2. Introduction to Lab 7: Ex. Preparation of Culture Media The purpose of this exercise is to introduce the concept of growing microorganisms in the lab in order to be able to study them. Culture is the term given to microorganisms that are cultivated in the lab for the purpose of studying them. Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth (that is, multiplication) in order to cultivate these microorganisms in large numbers to study them.

3. Among the different kinds of microorganisms the two groups that can be grown in cultures are bacteria and fungi. Algae and protozoa require many different nutrients in minute quantities that are difficult to anticipate and prepare in the lab. These organisms have different nutritional requirements and thus various kinds of culture media have been developed. Primary ingredients required by all living organisms include: a carbon source, water, minerals, and a nitrogen source.

4. Living cells need nutrients required for their structure (biosynthesis) as well as nutrients to provide them with energy to perform all of their various life processes. Nutrients are acquired from the environment in which they live in their natural habitat. Most of these nutrients are then processed within the cell through a variety of metabolic pathways to be incorporated in different ways. The process of building complex structures from simple building blocks is called anabolism. The process of breaking up complex materials to harvest the energy in them is called catabolism. The ability to use particular compounds is dependent upon the genetic makeup (DNA) that the cells have.

5. Since there are different kinds of organisms that can be grown in culture media with varying needs, culture media have also been formulated with different ingredients. Culture media may be found in one of three states: liquid (called broth) semi-solid solid. Media are solidified by the addition of solidifying agents such as agar (inert compound). Varying the concentration of agar will yield varying degrees of solidification.

6. Culture media may be classified as: Synthetic media (Defined) Complex (Non-synthetic) media Synthetic media contain only ingredients for which a complete chemical formula is known. Complex media contain at least one ingredient for which a chemical formula is not known (such as milk, egg, malt, animal tissues) Culture media can also be classified based on the function they perform in determining various characteristics of organism that are able to grow on/in them e.g. Differential, Selective media.

8. The ingredients in a medium will affect the chemical nature of the medium. This is important because organisms vary in their requirement for different environments. One such property is: pH (which is a measure of the amount of hydrogen ions in a particular medium). This has to be monitored during the preparation of media since this will influence the kind of organisms that are able to grow in the medium. The pH of the medium will thus determine which organisms are able to grow on the medium. For example, fungi prefer acidic media for their growth while bacteria grow on neutral pH media.

9. The primary function of culture media is to be able to grow particular organisms on/in them. It is important that these media are devoid of any other living organisms. This is possible through the process of sterilization (a process by which all living organisms and their spore forms are killed and the medium is made sterile) Culture media are most commonly sterilized through the process of autoclaving (using high temperatures that will kill all living organisms under increased pressure for specified periods of time – in an appliance called the autoclave)

11. -3 different culture media will be prepared in the class. -They will be sterilized in an autoclave and poured into Petri dishes. -These will then be used for the next exercise to be done in the lab. The 3 media that are to be prepared are: Dextrose Starch Agar (DSA) Malt Agar (MA) Nutrient Agar (NA) Be able to assign functions to each of the ingredients for any culture medium – that is identify the source of: carbon, nitrogen, minerals and any other specific function they may perform (such as solidification) Most common sources: - carbon are sugars (carbohydrates) - nitrogen are proteins (partially degraded – peptone) - animal tissue extracts as a source for minerals

12. For example: Nutrient Agar: Gelysate peptone- 5.0g (source of carbon and nitrogen); Beef extract - 3.0g (secondary source of carbon and nitrogen and primary source of minerals), Agar -15.0g (solidifying agent – if this were not added medium would be a broth) Aseptic Technique: These are various techniques that are used to minimize the introduction of microorganisms into media especially during transfer processes, such as – pouring of media into Petri dishes, inoculation of cultures These techniques include: cleaning the bench top work areas with disinfectant solution washing hands before starting work other specific techniques that will be demonstrated in the lab.

13. Aseptic Techniques …protective clothing …hand washing …bench cleaning …loop flaming …pipettors