Culturing a Micro-organism using Aseptic Technique

When a scientist is working with micro-organisms it is essential that they use the aseptic technique, as this:

Ensures that their culture of the micro-organism remains pure and therefore free of other micro-organisms

Unless aseptic culture techniques are followed strictly, an originally pure culture will definitely become contaminated with other unwanted species.

Micro-organisms are very useful in making a large number of food and drinks and usually grown in liquid broth or a solid medium such as agar and either in a flat dish called a petri dish, or as a slope in a narrow bottle.

Brewers use the yeast left over at the end of brewing to start their next brew. However, because of changes occurring to the yeast, they have to replace it with a pure yeast culture, about every ten brews. They will keep a pure culture of yeast in the refrigerator on an agar slope - we will culture it on a malt agar plate. Nutrient agar is often used in laboratories for growing micro-organisms.

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Agar is a jelly -like substance that melts when heated and sets when cooled, but does not contain any nutrients. The nutrients needed by the micro-organisms are moulded and dissolved into a molten agar. The nutrients agar sets when poured in a petri dish and allowed to cool.

Our aim: To culture micro-organisms using the aseptic technique with agar.

Safety points: before carrying out this experiment we had to make sure that we did it safely in the laboratory .We had to be very careful when dealing with the micro organisms and that we did everything quick so that no bacteria would get into the petri dish and we also had to wear safety goggles to prevent a unlikely event of an eye accident.

Food or drink should not be stored or consumed in a laboratory or prep room that is used for microbiology. No one should lick labels, apply cosmetics, chew gum, suck pens or pencils or smoke in a laboratory or prep room. Facilities should be provided within the laboratory or prep room for hands to be washed with soap after handling microbial cultures and whenever leaving the lab. Paper towels or some other hygienic method should be used for drying hands. If contamination of the hands is suspected, then they should be washed immediately with soap and water. Cuts or abrasions should be protected by the use of waterproof dressings or by wearing disposable gloves. Safety glasses may be worn, according to local requirements.

Materials and methods: For this experiment the materials we used were: a inoculating loop, fire resistant mat, Bunsen burner, goggles, an agar petri-plate with lid, bottle of yeast culture and a incubate to store the plate in. We also wiped down the bench with water to avoid contamination and culture the soil water. First of all we Placed are inoculating loop in a Bunsen flame for a few seconds until it glowed ash red and became increasingly hot. We then unscrewed the bottle containing the pure yeast culture, with the opposite hand [holding the inoculating loop] and we flamed the neck of the bottle. Soon after we inserted the sterile inoculating loop quickly into the bottle so as to pick up a film of the yeast culture in the loop.

As the loop is very hot we had left it in there for a few seconds to make certain we had picked up living cells. Then we took the loop out from the bottle and flamed the neck again just before sealing it. At this point we were hoping to have picked up a film of yeast culture on the loop. Now at this point we had to quickly and carefully open the lid of the agar plate and just sufficiently enough to insert the inoculating loop. We gently stroke the surface of the agar with the loop in the design of a zig-zag shape [called streaking a plate]. Then we took a flame to the inoculating loop to kill any yeast cells on it. After that the teacher had given us sticky labels to put our names and the date down with the type of organism [yeast]. And later we sealed the agar -plate with sellotape and incubate at 30?C for two days. Finally we saw our individual plate with the Colonies of yeast on the malt agar plate.

The result and evaluation: Unfortunately we got some contamination. This is the cause of bacteria coming into contact with the plate and the plate may have been open for too long or the equipment was not sterile. The need for a clean working environment when doing this experiment must be thought through carefully, for cleanliness is the prerequisite to any meaningful work. As demonstrated by the plate transfer process, many parts of the aseptic procedures require occasional exposure to the surrounding environment. Since our school laboratory cannot be made totally sterile, it is imperative that the room be kept clean.

I have found that it is especially important to work with a well characterized strain if the microorganism is used for food preparation, as well as in antibiotic production where the product is to be taken internally, and for the same reason that a student will not lick an incubated plate that was exposed to the air, the presence of a large number of contaminants may present a potential health hazard, especially when the exact natures of the contaminants are unknown.

My plate :

Agar is a jelly -like substance that melts when heated and sets when cooled, but does not contain any nutrients. The nutrients needed by the micro-organisms are moulded and dissolved into a molten agar. The nutrients agar sets when poured in a petri dish and allowed to cool. Agar is a compound in its own right. It is a polysaccharide found in the cell walls of some red algae and is unusual in containing sulfated galactose monomers. It requires nothing but extraction and purification to become agar, but is sometimes chemically modified into agarose for special applications. Agar added to media simply gels them into a convenient solid form.

I have done some investigating inside the brewing industry and what we did. I have found out that the recent amalgamation of a number of brewing companies and the continued increase in national and international franchise/contract brewing has resulted in brewing companies and individual breweries handling an ever-increasing number of yeast strains. Genetically stable, aseptic and confidential storage of a master culture of each yeast strain is essential. It is common practice for brewery yeast cultures to be used to pitch fermentations a maximum of ten times. Centrally held master cultures are used to supply fresh yeast for brewery propagation and subsequent fermentations.

The method of choice for long-term yeast strain storage is under liquid nitrogen, a method that has been used by Scottish Courage Brewing Ltd since 1983. Master cultures were selected from existing brewery yeast populations after undergoing a range of microbiological, biochemical and fermentation tests. Franchise partners have supplied other strains. The poster will describe the 'cascade' system used by Scottish Courage for the storage of its brewing yeast strains. Agar slopes (slants) made from master cultures are prepared and quality assured following ISO 9000 accredited methodology. Batches of approximately 20 slopes are made from each liquid nitrogen straw, and a sacrificed slope undergoes testing for microbiological contamination, viability and yeast mutants (petites).

In addition, the identity of each batch of slopes is confirmed using molecular biology analysis techniques such as PCR. Duplicate cultures of all yeast strains are held confidentially by the National Collection of Yeast Cultures as a back up. In all, twelve brewing yeast strain master cultures are held under liquid nitrogen at the [Scottish Courage Technical Center in Edinburgh] and are used to supply ten breweries with over 600 agar slopes between them on an annual basis. The yeast storage and supply management systems in place have proved to be robust and reliable over a number of years, giving the breweries in the group confidence in the quality of the yeast supplied to initiate brewery propagations.