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Isolation of pure culture took place on BIOLOG media; gram stain was then performed to illustrate the testing protocol. Inoculum is prepared at a specified cell density with the use of a turbid meter. Inoculation is done on a micro-plate, which is then read, and identity is determined.
The pure culture used for identification was tested by gram stain. We did this to determine if the organism was bacteria, gram-positive or negative. One must be very alert for variables in gram, which indicated the different microbes present. We realized those good techniques were of great significance, and mistakes in determining the shape and gram reaction could put an individual on the wrong track.
Aerobic bacteria’s Microplates were read after a period of four to six-hour incubation. The resulting pattern could be read manually with the naked eye or using a microplate reader at 590nm. White coverer was removed and placed on a white sheet. Dark purple wells meant positive while clear wells indicated negative.
The next step was manually entering the reactions into the onscreen microplate. A purple circle signified a positive result. Whereas white circles meant negative results, we individually changed the wells positive to negative by clicking on all positive.
The system identified the microorganisms by the use of extensive database software. The microplate indicated a good match with the organism an ID appeared in the bar area, which is green in color, at the middle of the screen which is at the pinnacle of the results area.
The software demonstrated microbes on a list that is ranked with the entry number one topping up, as the best matched chosen from the database. A (+) sign found in a negative well implied that the BIOLOG database was positive for that particular test. Gram-positive Rod was also used to determine if it was spore-forming bacillus, as special handling was required in spore formers as they often grow at a faster rate.
Directions and protocols must be followed meticulously, we discovered this can be a weakness, if not properly guarded. On the other hand, grams positive bacteria were a bit challenging to identify. It is mandatory to incubate anaerobic bacteria with a container together with a headspace of nitrogen. Some organisms are capable of producing nutrient-rich slime coating, and cells can actually feed on them, this, however, breeds false negatives.
It is imperative to ensure that the culture is first filtered into non-selective media. With this in place, all wells can stand positive. Use of proper aseptic techniques is always necessary. Disposable sterile plastic ware, as well as glassware, should be employed when handling cell solutions and suspensions. Glass cell that is already washed may have some traces of detergent which can affect the findings with sensitive strains.
Failure to use the right media, atmosphere, and temperature for incubation influences the accurateness of the database generated, which results in BUG media. Not following the correct protocol or cutting corners and using old sporulating or unhealthy cells to make a suspension. By using worn-out cells, the system tests the metabolic properties of the cells that are living because some of them are liable to losing their metabolic strength by subjecting the cells to stress, even for a short period.
BIOLOG 2011. Web.