In hospitals, biological labs, and other settings requiring aseptic practices, disinfectants and antiseptics frequently perform identical tasks and are utilized. The primary application of these chemicals is to eliminate undesirable infections and dangerous bacteria during laboratory processes. In order to achieve the necessary results, certain laboratory operations require special techniques. Similar to other labs, this one uses the Kirby Bauer Disk diffusion method to determine the microbial species' sensitivity to both antibiotics and antimicrobials. This technique is comparable to the filter paper disk method. The practice is of great significance the learner is equipped with the skills to determining the sensitivity of cultures of bacteria. In addition, different disinfectants or an antiseptic acts differently on different strains of bacterium which is one the critical issues of concern due to antibiotic selection.
Materials
Bacterial broth culture which was to provide suitable nutrients for bacterium multiplication (Staphylococcus saprophyticus and Escherichia coli)
4 TSA plates (trypticus soy agar)
Sterile filter paper
70% alcohol to sterilize the tools and equipment to be used such as
Sterile cotton swabs
forceps
Metric ruler
6 disinfectants
Procedures
The sides of the plate as well as the nutrient agar were examined carefully ensuring no bacterial colonies establishment. Humidity was another factor and no drops of water on the agar that was allowed. This was to ensure very minimal to no contamination on the experiment to be undertaken. A sterile cotton swab was used to dry the plates.
The bacterium culture was obtained by use of a sterile swab through gently swirling it around the culture. To remove excess liquid, the swab is pressed on the sides of the glass tube.
The plate was then inoculated through rubbing the swabs against the agar (TSA) to spread in three directions as illustrated below.
Zigzag lines were first made, the plate was then rotated to 450 in two rounds making a ‘bacteria lawn’
The plates were then allowed to sit for five minutes after the inoculation
Kirby-Bauer method
The bacterial lawns are prepared as illustrated above on each plate
The filter paper antibiotic disks are placed on the agar surface through the antibiotic dispenser. The dispensers are then keenly examined to ensure that each has a disk in the cartridge whereby after dispensing the green disk a replacement from the instructor was obtained.
The plates were placed in inverted position in an incubator at 37° Celsius. The figure below shows bacteria lawn created for testing antibiotic resistance.
Fig 2 showing bacteria lawn for testing resistance to antibiotic
Basic technique
The forceps are sterilized through alcohol flame sterilization. This is done through dipping the forceps in alcohol then gently burning it on a flame to dry of.
A permanent marker was then used to in dividing the TSA plate to six parts as shown below. Antiseptic disk are then placed into each of the quadrant.
Bacterial lawns were then prepared on each plate.
Figure 3 illustrating the markers on the six quadrants
A sterile filter paper was placed in the antiseptic provided using a sterile forceps which was then placed on the quadrant the antibiotic disk was then tapped gently in attempts of securing to the agar.
The process was then repeated using other five disinfectants. Forceps were sterilized each time a filter paper disk was obtained.
The plates were placed in an inverted position in the incubator at a temperature of 37°.
The petri dishes were then collected from the incubator where observation on the bacterial growth was done.
Observation on the clear zones was done and noted keenly and records taken.
We will email this sample to you with pleasure!
Type your email