BASIC BACTERIOLOGICAL TECHNIQUES

An experienced microbiologist employs many techniques and skills when handling microorganisms. More so than in any other field of science, the skills of a microbiologist need to be practiced and refined to achieve a level of proficiency. Microscopy is one skill you are already learning and will soon master with further practice. Other skills of the microbiologist allow for the safe handling and manipulation of microorganisms. Isolation and investigation of microorganisms are formidable tasks confronting both novice and experienced microbiologists. The skills employed to achieve these tasks are called aseptic technique.

Aseptic technique reduced the potential spread of bacteria used in laboratory to you or other people. This could happen if a pathogen were allowed to escape the lab on contaminated clothing, books, or other materials. You will be introduced this week to the basic skills of aseptic technique, and will be expected to apply and refine them over the course of the semester.

Aseptic technique is necessary to prevent contamination of pure cultures (a culture containing a single species) which are used routinely for analysis. Extraordinary precautions are needed to prevent contamination because microorganisms, particularly bacteria, are ‘everywhere’. Trillions of bacteria occur in, on and around our bodies, and even slight carelessness can lead to inadvertent contamination. The vast majority of these bacteria are harmless, even beneficial, but analysis of a contaminated culture will yield unpredictable and most likely erroneous results.

In Exercise III of this lab exercise, you will isolate an unknown bacterium, separate it from contaminants, and grow it as a pure culture. This will be the “semester unknown” that your team will maintain and analyze throughout the semester. Remarkably, when bacteria are collected from a site (such as by swabbing a surface) and transferred to standard bacterial culture media, only a small fraction of the cells present will actually grow. Those that do grow are abundant enough, but most species have specialized growth requirements that are not met by standard culture media.

Summary of exercise
I. You will practice making aseptic transfers between culture media.
II. You will practice making a streak plate using a provided mixed bacteria culture
III. You will inoculate a plate of nutrient medium from an environmental site to demonstrate the prevalence of microorganisms in the environment and use streak plating to isolate an unknown bacterial species.
IV. You will examine bacteria cultured on solid media to study the importance of colony morphology in identifying bacterial types.

I. Practicing Aseptic Transfers

The technique that you will use most frequently during the semester is aseptic transfer of bacteria, which you will do whenever you inoculate culture media with an organism. The inoculating loop (shown to the right) is the standard tool of the microbiologist and is used to transfer cells to and from culture media. An important point to remember is that seeing a mass of bacteria on the loop during a transfer is not necessary (although it is reassuring))even a single cell is adequate to start a new culture. An essential rule for any aseptic transfer is to sterilize the inoculating loop BEFORE and AFTER each transfer. Why? The techniques for doing aseptic transfers will be demonstrated in class. The manner in which you hold the tubes, caps and inoculating loop are important. You are encouraged to ask for assistance or a critique while you perform the transfers.

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Source: http://w3.marietta.edu/~spilatrs/biol202/labexercises/1-Basic_techniques.pdf

Purpose

     Recognition of media which used  for cultivaring microorganisms and their preparation and sterilization. Learning of to colony selection techniques with pure culture techniques and inoculation of the cultures to different media by transferring techniques.

Theory

      The Techniques of Pure Cultures  

     Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is onlyone microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation. Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are called disinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

Materials

·       Nutrient Agar

·       Nutrient broth

·       Autoclave

·       Beaker

·       Balon Tube

·       Distilled Water

·       Bunson Burner

·       Stirring magnet

·       Cotton

·       Rope

·       Flask

·       Test Tubes

·       Aliminium foil

·       Electrical heater

·       Sterile pipettes

·       Sterile petri dishes

·       Escherichia coli in Nutrient broth

·       Sterile Nutrient agar slant,plate and deep

·       Needle

·       İnoculation loop

·       Alcohol

·       Dilution tubes

Procedure

     Preparation of Nutrient broth: 3.2g of Nutrient broth media was taken and was placed in the beaker.A magnetic stirrer was added.After 400ml of distilled water was added in the beaker.İngredients was mixed.This mixture was distibuted into test tubes about 5-6ml for each tubes.Next, mouth of tubes were closed by

Results and Calculations

Discussion

İn this experiment we learned to prepare different media according to a certain microorganisms and a certain purposes. These media are anaerobic, synthetic, transport, enriched, selective, differential and microbiological assay media. For example; in anaerobic media; oxygen is removed from media with reducing agent, in synthetic media; their chemicals and concentration is known and identified, in transport media; microorganism is transferred from place to other place temporarily, in enriched media; number of scarce microorganisms are increased, but during this process growth of other microorganisms are not prevented, in selective media; growth of a special microorganisms is supplied and growth of other microorganisms are prevented, in differential media; appearance and size of microorganisms are determined with indicator, in microbiological assay media; concentration of some substances are measured. These media are prepared from nutrient broth and nutrient agar that broth represents liquid media and agar represents solid media.

Some media are slant media shape some media also are vertical shape. Slant media provide a large surface area so slant media is convenient for aerobic organisms, in vertical (deep) media, microorganism is cultivated toward the penetration of media and Petri plate also supplies a large surface area and because of this it supplies growth of microorganisms in a short time.

Finally; sterilization of media and equipment were learned during experiment. Moreover, required criteria one by one was told us. For sterilization, applied processes are classified as physical and chemical methods. Physical methods are heat, wet sterilization, tyndallization dry heat sterilization, radiation, freezing and bacteriological filtration. Chemical methods are salt, phenol and phenol derivatives, halogens, and alcohols. Chemical and physical methods and their effects on the microorganisms and equipment were learned and examined.

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; microorganisms were observed, another petri plate had been exposed to cough that a few microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.