Microorganisms and Bacteria: Aseptic Technique, Streak Plate Method

Microorganisms and Bacteria
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1 Aseptic Technique
Aseptic technique is a set of specific laboratory practices that are always performed before beginning experiments. The specific practices are meant to maintain as well as maximize the control and eradication of pathogens in the laboratory set up. They are always carried out under strict sterile conditions to either reduce or eliminate pathogenic contamination in the laboratory working areas. The aseptic technique achieves greater control or eradication of microbial contamination than other practices that focus on sanitization of microorganisms. In order to ensure complete control of pathogenic microorganisms, it is critical to follow the aseptic technique requirements to the letter. There are fundamental practices that should always start before carrying out any form of experiment. These practices include:
* Cleaning the laboratory working bench: The laboratory working bench should be cleaned thoroughly using either absolute ethanol or 70% ethanol. This ensures that any microorganism present is inactivated before a scientist begins an experiment.
* Flaming loops: This aseptic technique is done to inactivate any microorganisms that might be present on loops and straight wires, and that might interfere with a new experiment. Usually, loops and straight wires are introduced into a Bunsen burner flame for a few minutes until red hot. When the loops and wires glow red hot, this confirms that any unwanted microorganism is inactivated.
* Inoculation of agar slants: When introducing microorganisms’ cultures on agar slants or any other form of agar, wire loops should be used to inoculate. Caution, however, should be exercised in order to avoid breaking the solid agar. Inoculation should specifically be carried out in a zigzag manner.
2 Streak Plate Method
In the Streak Plate procedure, it is important to use a loop (either a nickel-chromium wire or disposable plastic) to transfer an inoculum onto a differential medium. Using the loops is among the critical practices required in the aseptic technique because they reduce the risk of contamination and also infection. It is also easier to sanitize the loops (wires and plastics) than it is with other materials.
The Streaking Plate procedure is carried out in a laminar flow hood in an aseptic technique as follows:
* Labeling petri dishes: The procedure begins with labeling of petri dishes to be used in inoculation. Usually, petri dishes are labeled on the bottom close to the edge of the bottom of the dish; this is done to reserve some space for observation after the dish has been incubated together with the inoculum. Labels must bear the inoculum name, the type of agar used, the date, and name or initials of the plater. If any water is observed on the agar, it should be removed using a sterile cotton swab.
* Sterilization of Loops: Even before obtaining a specimen of the inoculum, the transfer wire loops should be sterilized by using a Bunsen burner. The flame of the burner should heat the loop until it is red hot; this confirms inactivation of any microorganisms that might be present on the loop prior to beginning an experiment. The loop is then allowed to cool slowly usually by holding it in the air. After the loop has cooled, it can be used to transfer the inoculum onto the petri dish containing the agar. It is recommended that a new loop be used (in case of plastic loops) or be sterilized (in case of wire loops) when streaking each new sector on the petri dish.
* Streaking the Plate: When inoculating the agar, the lid of the petri dish is removed. The streaking process should be shorter to minimize exposure of agar to air. The first streak is applied on one area of the dish. By touching the loop on the agar, the inoculums on the loop are transferred onto the plate. Normally, the inoculums are applied in a zigzag manner, making sure that the loop movements are closer to each other. The loop should also glide over the surface of the agar and should not penetrate the agar. After the first streak, the loop should be removed from the dish and sterilized or replaced (in case of disposable plastic). Streaking on the second sector involves touching the loop on the first sector once and then streaking a second region of the dish with the inoculum obtained from the first region. However, streaking in the second sector should be done lightly than in the first. The third streaking process involves touching a sterilized loop onto the second sector and obtaining the inoculum. The inoculum is then spread onto a new region, different from the first and second regions. This sector should be streaked ensuring wider gaps are maintained than in the previous streaks. Care should also be taken not to touch the first streak sector when completing the streaking pattern in a quadrant model. The final step in this procedure involves the flaming of the wire loop in order to inactivate any inoculum left in the process. The inoculated petri dish should then be inverted; in this case, the agar side should face up. The petri dish can then be incubated to allow for isolation.
3 Escherichia coli:
The natural habitat for Escherichia coli is the gastrointestinal tract of human beings as well as other warm blooded animals. E. coli is actually the most abundant facultative anaerobe found in the human gastrointestinal tract (GIT). Though a significant number of E. coli strains do not cause harm in humans and animals, some strains are known to be very pathogenic. These pathogenic strains are known to cause a number of serious infections including bacteremia, urinary tract infections (UTIs), severe diarrhea as a result of food poisoning, neonatal meningitis, pneumonia, cholangitis, and cholecystitis.
As mentioned above, not all strains of E. coli are pathogenic; the ability to cause disease in the different available strains depends on the presence or absence of virulence factors. Virulence factors are special genes that code for different toxins that the E. coli releases upon contact with a host organism. Some strains possess the virulence factors while others do not. Most untrained microbiologists, including students, train with the non-pathogenic strains of E. coli in laboratory settings. This is because these strains do not have the ability to cause infections, and can therefore be manipulated easily than the pathogenic strains. This is especially so when referring to the aseptic technique. For such non-pathogenic strains, the aseptic technique can be easy to practice and handle without fear of being infected; however, the pathogenic strains require professionals to handle them because there is a greater risk of importing infections arising from unprofessional handling. E. coli plates should be incubated at 37ºC because that is the optimum temperature for the bacteria. The bacteria live in the gastro intestinal tract of humans and warm blooded animals. The optimum temperature in human beings is around 37ºC. At this temperature, the bacteria cannot be denatured; they show optimal growth.
4 Selective and Differential Growth Media
A selective medium usually allows the growth of a desired organism while stopping or killing non-desired organisms in the medium. On the other hand, a differential medium allows for the identification of the desired organism without killing non-desired organisms. The differential media contain useful nutrients that allow for clear identification of target organisms. The nutrients present usually take advantage of the organism’s biochemical properties to bring out an observable change when the target organism is present. On the MacConkey agar, Escherichia coli appear as flat, dry, and pink colonies with the surrounding area of the agar being dark pink in color. The bile salts normally present in the MacConkey agar are precipitated in these surrounding areas. The reason behind the formation of the pink E. coli colonies on the MacConkey agar is due to the fermentation of lactose. Lactose is one of the many components found in the MacConkey agar. E. coli colonies are known to ferment lactose to produce lactic acid. The acid is responsible for the drop in pH of the agar and the pink color of the colonies.
5 Contamination
MacConkey agar is a selective as well as a differential medium. However, it does not allow the growth of gram-positive bacteria and fastidious gram-negative bacteria including Pasteurella and Neisseria. For this reason, MacConkey agar cannot allow more contaminants compared to other nutrient agar. The crystal violet dye and bile salts present in MacConkey agar prevent the growth of the above unwanted bacteria. However, MacConkey agar has its limitations too. For example, the agar can allow for the growth of some gram-positive strains of bacteria without complete inhibition. Some enteric gram-negative bacilli strains may grow very slowly or not grow at all on selective media. This may affect interpretation of results. Another li...